WO2022244809A1 - Recombinant microorganism having diamine producing ability and method for manufacturing diamine - Google Patents

Recombinant microorganism having diamine producing ability and method for manufacturing diamine Download PDF

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WO2022244809A1
WO2022244809A1 PCT/JP2022/020689 JP2022020689W WO2022244809A1 WO 2022244809 A1 WO2022244809 A1 WO 2022244809A1 JP 2022020689 W JP2022020689 W JP 2022020689W WO 2022244809 A1 WO2022244809 A1 WO 2022244809A1
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diamine
recombinant microorganism
culture
microorganism
compound
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PCT/JP2022/020689
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French (fr)
Japanese (ja)
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令 宮武
章友 牛木
光二 井阪
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旭化成株式会社
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Priority to JP2023522698A priority Critical patent/JPWO2022244809A1/ja
Priority to EP22804720.5A priority patent/EP4353814A1/en
Priority to CN202280035194.1A priority patent/CN117500912A/en
Publication of WO2022244809A1 publication Critical patent/WO2022244809A1/en

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds

Definitions

  • the present invention relates to a halophilic and/or alkalophilic recombinant microorganism capable of producing diamine, which is an industrially useful compound, and a method for producing diamine using the recombinant microorganism.
  • the present invention also relates to halophilic and/or alkalophilic recombinant microorganisms with suppressed N-acetyldiamine production.
  • Diamine along with dicarboxylic acids such as adipic acid, is an industrially important compound as a raw material for polyamide (PA66 (6,6-nylon), etc.).
  • 1,6-hexamethylenediamine also called 1,6-diaminohexane or hexamethylenediamine, which is a compound having the molecular formula C 6 H 12 N 2
  • PA66 nylon 66
  • Hexamethylenediamine is also used as an intermediate for urethane raw materials, agricultural chemicals, and pharmaceuticals via isocyanate.
  • Hexamethylenediamine is synthesized by hydrocyanation of butadiene, electrolytic dimerization of acrylonitrile, or nitrilation of adipic acid to obtain adiponitrile, which is then hydrogenated using a catalyst such as nickel (Non-Patent Document 1). Although hexamethylenediamine is industrially produced by this method, once adiponitrile is synthesized, a hydrogenation reaction is carried out.
  • Patent Document 1 As a method for producing hexamethylenediamine using microorganisms, a method has been reported in which diamine is produced from intracellular dicarboxylic acids, aminocarboxylic acids, and dialdehydes by combining exogenous enzymes such as carboxylic acid decarboxylase and aminotransferase.
  • Patent Document 1 In Patent Document 1, in a host microorganism modified to have a hexamethylenediamine production pathway, an enzyme gene whose yield is expected to be improved by deletion and/or disruption is predicted based on an in silico metabolic simulation, exemplified.
  • Patent Document 2 describes a method for producing hexamethylenediamine by an enzymatic reaction pathway via 6-hydroxyhexanoic acid. However, it does not refer to the generation of by-products derived from intermediates in the hexamethylenediamine production pathway newly constructed by genetic recombination, and a method for suppressing them.
  • 1,5-Pentamethylenediamine also called 1,5-diaminopentane or cadaverine, a compound with the molecular formula C 5 H 14 N 2
  • PA56 fiber has the same strength and heat resistance as PA66, and exhibits high hygroscopicity and high hygroscopicity, so new market development is expected.
  • 1,5-Pentamethylenediamine is also attracting attention as a raw material for urethane via isocyanate, an intermediate for agricultural chemicals and pharmaceuticals.
  • Non-Patent Document 2 While an efficient chemical synthesis method from petrochemical raw materials of 1,5-pentamethylenediamine has not yet been established, it is known to be easily produced by enzymatic decarboxylation of L-lysine in biosynthesis.
  • PA56 resin which is made from biomass-derived 1,5-pentamethylenediamine, has been attracting industrial attention as one of bioplastics from the viewpoint of reducing environmental load (Non-Patent Document 2).
  • fermentation-producible amine compounds include 1,5-pentamethylenediamine and hexamethylenediamine, which can be used as raw materials for polymer production in the production of chemical products (Patent Documents 3 to 7 and Non-Patent Document 3).
  • Patent Document 8 As a method for producing 1,5-pentamethylenediamine using microorganisms, E. coli in which lysine decarboxylase is highly expressed is cultured, and the precursor lysine is reacted with the enzyme to decarboxylate 1,5-pentamethylenediamine. A method for obtaining methylenediamine is known (Patent Document 8). There is also a method of producing 1,5-pentamethylenediamine from glucose by culturing coryneform bacteria in which the activity of the lysine decarboxylase gene is increased and the activity of the gene that plays an important role in lysine biosynthesis is decreased. It has been proposed (Patent Document 9).
  • Patent Documents 10, 11 and 12 As a method for separating and purifying diamine from the culture medium, for example, an alkaline solution such as sodium hydroxide is added to liberate 1,5-pentamethylenediamine, and then an appropriate solvent is used for extraction. known (Patent Documents 10, 11 and 12). A method has also been proposed in which an aqueous solution of 1,5-pentamethylenediamine carbonate is thermally decomposed to separate crude 1,5-pentamethylenediamine and carbon dioxide, and then the diamine is purified by distillation (Patent Document 13). .
  • an alkaline solution such as sodium hydroxide
  • Patent Documents 11 and 14 use polar organic solvents such as chloroform and hexane for extraction, but many organic solvents are harmful and should be handled properly. I don't like it.
  • the low extraction efficiency greatly affects the production cost.
  • the used organic solvent is recovered in order to reduce the production cost, the production process becomes complicated.
  • purification is performed by a crystallization method, but the crystallization rate is 40 to 45%, and a high yield cannot be expected.
  • a large amount of salt is generated as a by-product in the diamine liberation step.
  • a maximum of about 100 g/L of sodium sulfate is generated as a by-product due to the presence of sulfate ions, which are counter ions of cadaverine.
  • the method described in Patent Document 14 does not mention the handling of the aqueous phase after diamine solvent extraction, which contains inorganic salts, but this method may also require a great deal of cost for waste liquid treatment. .
  • an object of the present invention is to provide a recombinant microorganism and a diamine production method that can achieve at least one of process simplification and wastewater treatment cost reduction in the production of diamine.
  • a further object of the present invention is to provide a halophilic and/or alkalophilic recombinant microorganism in which N-acetyldiamine production is suppressed.
  • the inventors conducted extensive studies and found that the host microorganism of the present invention, which has at least one of halophilicity and alkalophilicity, is modified to have diamine-producing ability. It has been found that recombinant microorganisms can achieve at least one of process simplification and wastewater treatment cost reduction in diamine production.
  • the present invention provides: [1] A halophilic and/or alkalophilic recombinant microorganism having diamine-producing ability,
  • the diamine is represented by the formula: NH 2 CH 2 (CH 2 ) n CH 2 NH 2 (wherein n is an integer from 0 to 10), A recombinant microorganism in which a halophilic and/or alkalophilic host microorganism has been modified to have diamine-producing ability;
  • the genetic manipulation includes the following (A), (B), (C), (D) and (E): (A) an operation of introducing an exogenous gene encoding the L-lys
  • the mutagenesis or genetic recombination is performed by at least aspartokin
  • the present inventors conducted intensive studies in order to solve the above further problems. Specifically, the present inventors discovered the use of halophilic and/or alkalophilic recombinant microorganisms to simplify the diamine production process and reduce wastewater treatment costs, as described above, and By using a halophilic and/or alkalophilic recombinant microorganism as a microorganism, simplification of the pH control step during the production of 1,5-pentamethylenediamine and the liberation step of 1,5-pentamethylenediamine.
  • 1,5-pentamethylenediamine by halophilic and/or alkalophilic recombinant microorganisms
  • 1,5-pentamethylenediamine N-acetylcadaverine, in which the N-terminal amino group of methylenediamine was acetylated, was produced as a by-product.
  • N-acetylation of 1,5-pentamethylenediamine is catalyzed by an acetyltransferase endogenous to microbial cells.
  • an N-acetylation enzyme of 1,5-pentamethylenediamine has been identified, and the N-acetylation of 1,5-pentamethylenediamine can be suppressed by disrupting the gene encoding the enzyme.
  • Patent Document 9 and Japanese Patent No. 5960604 Japanese Patent No.
  • the present inventors determined to identify a gene encoding an enzyme that catalyzes the acetylation of 1,5-pentamethylenediamine from the genome sequences of halophilic and/or alkalophilic host microorganisms. Successfully, modifications have been made to the gene leading to suppression of by-product N-acetyldiamine production in halophilic and/or alkalophilic host microorganisms.
  • the present invention further provides: [26] A halophilic and/or alkalophilic recombinant microorganism capable of producing diamine, A recombinant microorganism comprising one or more genetic modifications that inhibit an N-acetyltransferase that N-acetylates a diamine compound to produce an N-acetyldiamine compound; [27] The genetic modification is - modification that suppresses the expression of the endogenous gene encoding the N-acetyltransferase, or - The recombinant microorganism of [26], which is a modification that reduces the activity of the N-acetyltransferase; [28] according to [26] or [27], wherein the ability to produce an N-acetyldiamine compound is suppressed or eliminated compared to the production ability of the non-mutant strain that does not contain the genetic modification; a recombinant microorganism of [29] The recombinant microorganism
  • (A-3) 1 to 10, 1 to 7, 1 to 5 relative to the amino acid sequence shown in SEQ ID NO: 23 consisting of an amino acid sequence in which 1 or 1 to 3 amino acids are deleted, substituted, inserted and/or added, and having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound, or (B) (B-1) DNA consisting of the base sequence shown in SEQ ID NO: 24; (B-2) an enzyme that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 24 and N-acetylates a diamine compound to produce an N-acetyldiamine compound DNA encoding a protein having activity; (B-3) consists of a base sequence having 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the base sequence shown in SEQ ID NO: 24, and DNA en
  • At least one of process simplification and wastewater treatment cost reduction can be realized in diamine production.
  • N-acetyldiamine which is a by-product
  • the production of N-acetyldiamine can be suppressed in diamine production using halophilic and/or alkalophilic diamine-producing bacteria.
  • FIG. 1 is a diagram showing an example of a hexamethylenediamine production pathway.
  • FIG. 2A shows the amino acid sequences of each enzyme.
  • FIG. 2B is a diagram showing the amino acid sequence of each enzyme.
  • FIG. 3 shows the amino acid sequence (SEQ ID NO: 23) of the yjbC enzyme of Bacillus pseudofirmus.
  • FIG. 4 shows the nucleotide sequence (SEQ ID NO: 24) of the Bacillus pseudofirmus yjbC gene.
  • FIG. 5A is a diagram showing base sequences of primers.
  • FIG. 5B is a diagram showing base sequences of primers.
  • a numerical range indicated using "-" indicates a range that includes the numerical values before and after "-" as the minimum and maximum values, respectively.
  • the upper limit value or lower limit value of the numerical range in one step can be arbitrarily combined with the upper limit value or lower limit of the numerical range in another step.
  • endogenous or “endogenous” means that a host microorganism that has not been modified by genetic recombination has the gene referred to or the protein (typically an enzyme) encoded by it. , is used to mean that the host microorganism has whether or not it is functionally expressed to the extent that it can drive dominant biochemical reactions within the host cell.
  • endogenous and endogenous are used interchangeably herein.
  • the term “foreign” or “exogenous” is used when the host microorganism before genetic recombination does not have the gene to be introduced, when the enzyme by the gene is not substantially expressed , and to denote the introduction of a gene or nucleic acid sequence into a host where a different gene encodes the amino acid sequence of the enzyme but does not express comparable endogenous enzymatic activity after genetic recombination .
  • the terms “exogenous” and “exogenous” are used interchangeably herein.
  • the culture solution obtained by culturing the microorganism may contain a diamine, or a diamine precursor such as a carboxylic acid compound, an aldehyde compound and/or a carbonyl compound is added to the medium, Diamines may be produced by culturing microorganisms that convert diamine precursors to diamines.
  • a "microorganism capable of producing diamines” includes microorganisms having one or more of these properties.
  • a microorganism has a diamine production pathway.
  • a microorganism “has a production pathway” means that the microorganism expresses a sufficient amount of an enzyme for each reaction step in the production pathway of the compound to proceed, and produces the compound. It means that it can be biosynthesized.
  • the recombinant microorganism of the present invention may be one using a host microorganism that originally has the ability to produce diamine, or a halophilic and/or alkalophilic host that does not originally have the ability to produce diamine. Microorganisms that have been modified to have diamine-producing ability may also be used.
  • diamine compounds are represented by the formula : NH2CH2 ( CH2 ) nCH2NH2 .
  • n is, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 2, 3, 4 or 5, more preferably 2, 3 or 4, particularly preferably is 3 or 4.
  • diamine compounds examples include ethylenediamine, propylenediamine, tetramethylenediamine (eg putrescine), pentamethylenediamine (eg cadaverine), hexamethylenediamine, and heptamethylenediamine.
  • the diamine compound is hexamethylenediamine or cadaverine.
  • Invention A Recombinant microorganism having diamine-producing ability and method for producing diamine A host microorganism having one side is modified so as to have diamine-producing ability.
  • the halophilic microorganisms described above can be used for the production of diamine, the water containing high concentrations of salts generated in the separation process can be reused as a medium. There have been no reports of applications of microorganisms having at least one of halophilicity and alkalophilicity to diamine production.
  • the present inventors used a microorganism having at least one of halophilic and alkalophilic properties as a host microorganism and attempted to modify it so as to have diamine-producing ability. According to the obtained recombinant microorganism, It has been found that at least one of process simplification and wastewater treatment cost reduction is possible.
  • the present inventors have succeeded in imparting diamine-producing ability to halophilic and/or alkalophilic microorganisms, and by using such microorganisms, pH Even when the pH is increased, the microorganism can be grown without adjusting the pH such as adding an acid solution, and in a more preferred embodiment, the high-concentration salt-containing water generated when isolating the diamine is used as a medium. We have found that it can be reused.
  • any foreign gene in the genome sequence Techniques for introducing sequences or removing unwanted gene sequences from genomic sequences can be used.
  • a technique of transforming a host microorganism having halophilicity, alkalophilicity, or both properties by combining a promoter capable of being expressed in the host cell and a gene involved in diamine production (2) The technology of (1) above, wherein the prior technology uses a plasmid vector that is stably replicated in cells of microorganisms having halophilicity, alkalophilicity, or both properties; (2') The technology of (2) above, wherein the plasmid vector to be the subject of the prior technology is pUB110, (3) The technology of (1) above, wherein the prior art is characterized by stably expressing desired traits in cells of microorganisms having halophilicity, alkalophilicity, or both properties; (4) Techniques for arbitrarily modifying gene sequences on chromosomes by combining temperature-sensitive plasmid vectors and negative selection, (5) The technology of (4) above, wherein the prior art stops replication of the plasmid carried by the micro
  • the prior art selects (negative selection) microorganisms that do not possess a specific gene by inhibiting the growth of microorganisms that possess a specific gene in the presence of substances such as sucrose and 4-chlorophenylalanine.
  • pheS gene host-derived phenylalanine tRNA synthetase ⁇ -subunit gene
  • a plasmid vector carries a high-expression promoter and a foreign gene, and is introduced into the cells of microorganisms having halophilicity, alkalophilicity, or both, thereby exposing the foreign gene to is stably highly expressed.
  • genome sequences possessed by halophilic, alkalophilic, or both halophilic and/or alkalophilic microorganisms are optionally edited using a combination of temperature-sensitive plasmid vectors and negative selection.
  • Cell lines whose gene transfer or genome sequence is edited using this technology may be wild-type strains in the usual sense, or auxotrophic mutant strains derived from the wild-type strains, antibiotics It may be a substance-resistant mutant strain. Furthermore, cell lines that can be used as host cells of the present invention may already be transformed to have various marker genes for mutations as described above. These techniques can provide beneficial properties for the production, maintenance and/or management of the recombinant microorganisms of the invention.
  • alkalophilic microorganisms that can be used as host microorganisms are a type of extremophilic microorganisms that exhibit diverse distributions, and are a general term for microorganisms that can grow even in an environment with a pH of 9 or higher. These are classified into obligate alkalophilic microorganisms that can grow only in an environment of pH 9 or higher, and facultative alkalophilic microorganisms that can grow at a pH of less than 9, although the optimum growth pH is at pH 9 or higher. Some of them can grow even in a strong alkaline environment with a pH of 12 or higher. All of these are alkalophilic microorganisms of the present invention.
  • halophilic microorganisms that can be used as host microorganisms is a general term for microorganisms that can cope with high-concentration salt stress.
  • non-halophilic bacteria whose optimum growth salt concentration is 0-0.2M sodium chloride, and low halophilic bacteria whose optimum growth salt concentration is 0.2-0.5M.
  • Moderate halophiles whose optimum growth salt concentration is 0.5 to 2.5M, and high halophiles whose optimum growth salt concentration is 2.5 to 5.2M. All of these except non-halophilic bacteria are halophilic microorganisms of the present invention.
  • Halophilic, alkalophilic, or both microorganisms that can be used as host microorganisms of the present invention include various microorganisms, non-limiting examples of which include Bacillus, Halomonas, Halo Bacteroides, Salinibacter, Alkalinephilus, Clostridium, Anaeroblanca, and the like.
  • the host microorganism of the invention is preferably a bacterium of the genus Bacillus. Among microorganisms belonging to the genus Bacillus, Bacillus pseudofirmus, Bacillus halodurans, and Bacillus marmarensis are preferred, and Bacillus pseudofirmus is more preferred.
  • the host microorganism is modified to construct a new diamine biosynthetic pathway.
  • the recombinant microorganism uses one or more L-lysine decarboxylase (EC 4 .1.1.18) obtained by introducing the enzyme gene into the cells of the host microorganism.
  • L-lysine decarboxylase (EC 4.1.1.18) is an enzyme that catalyzes the reaction of decarboxylating L-lysine to produce 1,5-pentanediamine.
  • a recombinant microorganism is obtained by inserting the enzyme gene sequence into the genome sequence of the host microorganism.
  • Such manipulation induces overproduction of L-lysine decarboxylase (EC 4.1.1.18) in the resulting recombinant microorganism. That is, the recombinant microorganism according to the present invention is modified by one or more genetic manipulations so as to induce overproduction of L-lysine decarboxylase (EC 4.1.1.18).
  • the genetic manipulation is, for example, one or more selected from the group consisting of (A), (B), (C), (D) and (E) below. It may be genetically engineered.
  • A an operation of introducing an exogenous gene encoding L-lysine decarboxylase into a host microorganism;
  • B an operation that increases the copy number of the endogenous gene for L-lysine decarboxylase in the host microorganism;
  • C an operation of introducing a mutation into the expression control region of the endogenous gene for L-lysine decarboxylase in the host microorganism;
  • D replacement of the expression regulatory region of the endogenous gene of L-lysine decarboxylase in the host microorganism with an exogenous regulatory region capable of high expression; and
  • E endogenous L-lysine decarboxylase in the host microorganism.
  • genes for L-lysine decarboxylase include cadA and ldcC of Escherichia coli.
  • the amino acid sequence of E. coli cadA enzyme is shown in SEQ ID NO:1, and the base sequence of E. coli cadA is shown in SEQ ID NO:2.
  • the amino acid sequence of E. coli ldcC enzyme is shown in SEQ ID NO:3, and the base sequence of E. coli ldcC is shown in SEQ ID NO:4.
  • the host microorganism is modified to construct a new diamine biosynthetic pathway.
  • a recombinant microorganism is obtained by introducing one or more enzyme genes into the cells of the host microorganism to construct a new hexamethylenediamine biosynthetic pathway.
  • a recombinant microorganism is obtained by inserting the enzyme gene sequence into the genome sequence of the host microorganism.
  • FIG. 1 shows an example of a hexamethylenediamine production pathway that microorganisms can have.
  • succinyl-CoA acetyl-CoA acyltransferase or 3-oxoadipyl-CoA thiolase
  • succinyl-CoA and acetyl-CoA are condensed and converted to 3-oxoadipyl-CoA.
  • enzymes that can catalyze this conversion include ⁇ -ketothiolase.
  • EC 2.3.1.9 (acetoacetyl-CoA thiolase) and EC 2.3.1.16 (3-ketoacyl-CoA thiolase), EC 2.3.1.174 (3-oxoadipyl-CoA thiolase) can be exemplified as enzymes that may have activity for this conversion.
  • the enzyme used in the present invention is not limited as long as it has activity for this conversion.
  • E. coli-derived PaaJ consisting of the amino acid sequence set forth in SEQ ID NO: 12 is used (Fig. 2).
  • 3-oxoadipyl-CoA is converted to 3-hydroxyadipyl-CoA.
  • enzymes that can catalyze this conversion include oxidoreductases classified in group EC 1.1.1.
  • Enzymes can be exemplified as enzymes that may have activity for this conversion.
  • the enzyme used in the present invention is not limited as long as it has activity for this conversion.
  • Escherichia coli-derived PaaH consisting of the amino acid sequence set forth in SEQ ID NO: 13 is used (Fig. 2).
  • 3-hydroxyadipyl-CoA is converted to 2,3-dehydroadipyl-CoA.
  • enzymes capable of catalyzing this conversion include hydrolyases classified in group EC 4.2.1.
  • EC 4.2.1.17 enoyl-CoA hydratase
  • EC 4.2.1.55 3-hydroxybutanoyl-CoA dehydratase
  • EC 4.2.1.74 long chain enoyl-CoA Enzymes classified into groups such as hydratases
  • the enzyme used in the present invention is not limited as long as it has activity for this conversion.
  • E. coli-derived PaaF consisting of the amino acid sequence shown in SEQ ID NO: 14 is used (Fig. 2).
  • step D conversion (2,3-dehydroadipyl-CoA reductase) of FIG. 1
  • 2,3-dehydroadipyl-CoA is converted to adipyl-CoA.
  • enzymes that can catalyze this conversion include oxidoreductases that fall into the group of EC 1.3.1.
  • EC 1.3.1.8 (acyl-CoA dehydrogenase (NADP + )
  • EC 1.3.1.9 (enoyl-ACP reductase (NADH)
  • EC 1.3.1.38 trans- 2-enoyl-CoA reductase (NADP + )
  • EC 1.3.1.44 trans-2-enoyl-CoA reductase (NAD + )
  • EC 1.3.1.86 crotonyl-CoA reductase
  • Enzymes falling into the groups EC 1.3.1.93 long-chain acyl-CoA reductase
  • EC 1.3.1.104 enoyl-ACP reductase (NADPH) have activity towards this transformation. It can be exemplified as possible enzymes.
  • the enzyme derived from Chaetomium thermophilum consisting of the amino acid sequence set forth in SEQ ID NO: 16
  • the Candida tropicalis consisting of the amino acid sequence set forth in SEQ ID NO: 17 Enzymes from A. are used (Fig. 2).
  • adipyl-CoA is converted to adipic acid.
  • enzymes that can catalyze this conversion include thioester hydratases that are classified in group EC 3.1.2.
  • enzymes falling into the groups EC 3.1.2.1 (acetyl-CoA hydratase) and EC 3.1.2.20 (acyl-CoA hydratase) may have activity for this conversion. It can be exemplified as an enzyme.
  • CoA-transferase Another example of an enzyme that can catalyze the conversion of step E in FIG. 1 is CoA-transferase, which is classified in the group EC 2.8.3.
  • EC 2.8.3.5 (3-oxoacid CoA-transferase)
  • EC 2.8.3.6 (3-oxoadipate CoA-transferase)
  • EC 2.8.3.18 succinyl- Enzymes classified into groups such as CoA:acetate (CoA-transferase) can be exemplified as enzymes that may have activity for this conversion.
  • acyltransferases include enzymes classified into groups such as EC 2.3.1.8 (phosphate acetyltransferase) and EC 2.3.1.19 (phosphate butyryltransferase).
  • Enzymes classified into groups such as EC 2.7.2.1 (acetate kinase) and EC 2.7.2.7 (butanoate kinase) are exemplified as enzymes that can have activity for this conversion. can be done.
  • adipyl-CoA is converted to adipic acid semialdehyde.
  • enzymes that can catalyze this conversion include enzymes classified in group EC 1.2.1.
  • EC 1.2.1.10 acetaldehyde dehydrogenase (acetylation)
  • EC 1.2.1.17 glyoxylate dehydrogenase (acylation)
  • EC 1.2.1.42 hexadecanal dehydrogenase (acylation)
  • EC 1.2.1.44 cinnamoyl-CoA reductase (acylation)
  • EC 1.2.1.75 malonyl-CoA reductase (malonic semialdehyde formation)
  • EC 1.2.1.10 acetaldehyde dehydrogenase (acetylation)
  • EC 1.2.1.17 glyoxylate dehydrogenase (acylation)
  • EC 1.2.1.42 hexadecanal dehydrogenase (acylation)
  • Enzymes classified into groups such as 2.1.76 succinic semialdehyde dehydrogenase (acylation) catalyze conversion reactions that eliminate CoA and generate aldehydes, similar to this conversion. It can also be exemplified as an enzyme that can have activity against The enzyme used in the present invention is not limited as long as it has activity for this conversion.
  • 2.1.76 succinic semialdehyde dehydrogenase (acylation)
  • the carboxyl group is transformed into an aldehyde.
  • Enzymes that can catalyze this conversion include, for example, carboxylic acid reductase (CAR).
  • CAR carboxylic acid reductase
  • EC 1.2.1.30 carboxylic acid reductase (NADP + )
  • EC 1.2.1.31 L-aminoadipate semialdehyde dehydrogenase
  • EC 1.2.1.95 L- 2-aminoadipate reductase
  • EC 1.2.99.6 carboxylic acid reductase catalyzes the conversion reaction of carboxylic acid to aldehyde in the same manner as this conversion.
  • carboxylic acid reductase can be converted to an active holoenzyme by phosphopantetheinylation (Venkitasubramanian et al., Journal of Biological Chemistry, Vol. 282, No. 1, 478-485 (2007) ).
  • Phosphopantetheinylation is catalyzed by phosphopantetheinyl transferase (PT).
  • Enzymes that can catalyze this reaction include, for example, enzymes classified under EC 2.7.8.7. Accordingly, the microorganism of the invention may be further modified to increase the activity of phosphopantetheinyltransferase.
  • Methods of increasing the activity of phosphopantetheinyl transferase include a method of introducing an exogenous phosphopantetheinyl transferase gene and a method of enhancing expression of an endogenous phosphopantetheinyl transferase gene. include, but are not limited to.
  • Enzymes used in the present invention are not limited to these as long as they have phosphopantetheinyl group transfer activity. et al., Journal of Biological Chemistry, Vol. 282, No. 1, 478-485 (2007)) and Lys5 of Saccharomyces cerevisiae (Ehmann et al., Biochemistry 38.19 (1999): 16171-6). mentioned.
  • the enzyme used in the present invention is not limited as long as it has activity for this conversion. For example, Npt derived from Nocardia iowensis consisting of the amino acid sequence set forth in SEQ ID NO: 21 is used (Fig. 2).
  • the transformations in steps J, M, P, and R in Figure 1 are transamination reactions.
  • Examples of enzymes that can catalyze this conversion include the transaminases (aminotransferases) that fall into the group of EC 2.6.1.
  • EC 2.6.1.19 (4-aminobutanoic acid-2-oxoglutarate transaminase) and EC 2.6.1.29 (diamine transaminase)
  • EC 2.6.1.48 (5-aminovalerate Enzymes classified into groups such as transaminase) can be exemplified as enzymes that may also have activity for this conversion.
  • the enzyme used in the present invention is not particularly limited as long as it has the conversion activity of each step.
  • the enzyme used in the present invention is not limited as long as it has activity for this conversion.
  • an E. coli-derived enzyme YgjG consisting of the amino acid sequence set forth in SEQ ID NO: 22 may be used (Fig. 2 ).
  • Typical amino group donors include, but are not limited to, L-glutamic acid, L-alanine, glycine.
  • Genes encoding the above enzymes that can be used in the present invention may be derived from microorganisms other than the exemplified microorganisms, or may be artificially synthesized, and substantially Any material can be used as long as it can express enzymatic activity.
  • the enzyme gene that can be used for the purpose of the present invention includes all mutations that can occur in nature, as well as artificially introduced enzyme genes, as long as they can express substantial enzyme activity in the host microbial cells. It may have variations and modifications. For example, it is known that there are extra codons for various codons that code for specific amino acids. Therefore, also in the present invention, alternative codons that are ultimately translated into the same amino acid may be used. That is, because the genetic code is degenerate, multiple codons can be used to encode a particular amino acid, such that an amino acid sequence can be encoded by any set of similar DNA oligonucleotides.
  • the recombinant microorganism has 85, 90, 92, 95, 98 or 99% or more homology with the nucleotide sequence shown in SEQ ID NO: 2 or 4,
  • the recombinant microorganism contains a nucleotide sequence having 92, 95, 98 or 99% or more homology, or the recombinant microorganism has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 3 and 85, 90, 92, It includes nucleotide sequences with 95, 98 or 99% or more homology, preferably 92, 95, 98 or 99% or more homology.
  • the above-mentioned diamine synthase gene as an "expression cassette” into host microbial cells, stable and high-level enzymatic activity can be obtained.
  • the gene sequence of the "expression cassette” is inserted into the genome sequence of the host microorganism to obtain more stable and high-level enzymatic activity.
  • an expression cassette means a nucleotide containing a nucleic acid sequence that regulates transcription and translation, functionally linked to a nucleic acid to be expressed or a gene to be expressed.
  • an expression cassette of the invention comprises a promoter sequence 5′ upstream from the coding sequence, a terminator sequence 3′ downstream, and optionally further conventional regulatory elements operably linked, such that In some cases, the nucleic acid to be expressed or the gene to be expressed is introduced into the host microorganism.
  • a promoter is defined as a DNA sequence that causes RNA polymerase to bind to DNA and initiate RNA synthesis, regardless of whether it is a constitutive promoter or an inducible promoter.
  • a strong promoter is a promoter that initiates mRNA synthesis at a high frequency, and is also preferably used in the present invention. For example, in Bacillus pseudofilamus, S-Layer protein synthetase, sigma factor (eg, rpoD), glycolytic enzyme (eg, glyceraldehyde-3-phosphate dehydrogenase), lactate dehydrogenase, glutamic acid A promoter region or the like for decarboxylase A is available.
  • the expression cassette described above is incorporated into a vector consisting of, for example, a plasmid, phage, transposon, IS element, phasmid, cosmid, or linear or circular DNA, and introduced into a host microorganism. Plasmids and phages are preferred in the present invention. These vectors may replicate autonomously in host microorganisms, or may replicate by being inserted into chromosomes. Suitable plasmids include, for example, pUB110, pC194 and pBD214 for bacilli such as Bacillus.
  • the expression cassette described above is preferably inserted into the chromosome compared to plasmids and phages.
  • Some selective pressure is required to maintain the plasmid within the host microorganism, generally requiring the addition of an antibiotic to the culture medium corresponding to the antibiotic resistance marker carried by the plasmid.
  • the gene expressed on the plasmid is unnecessary or burdens the growth of the host microorganism, the function of the enzyme endogenously retained by the host microorganism will cause mutation on the gene. There is a possibility that they may be introduced or deleted, and in many cases stable production of substances becomes difficult.
  • plasmids and the like include those described in "Cloning Vectors", Elsevier, 1985.
  • Introduction of an expression cassette into a vector can be performed by conventional methods including fragment amplification by PCR, excision with appropriate restriction enzymes, cloning, and various types of ligation.
  • a foreign gene can be introduced into the genome sequence of the host microorganism. , or unwanted genes can be removed.
  • the temperature sensitive origin of replication sequence is the origin of replication used in the pE194ts vector and the negative selection gene is the sacB gene or the mutated pheS gene.
  • the culture temperature is 37 to 43° C. or higher, that the medium contains 10% or higher sucrose, or that the medium contains 1 mM or higher 4-chlorophenylalanine. preferably.
  • the host microorganism is further modified by mutation or genetic recombination to improve lysine-producing ability. That is, the resulting recombinant microorganism further contains modification by mutagenesis or gene recombination to improve lysine-producing ability.
  • the mutation manipulation or the genetic recombination manipulation is, for example, at least one of aspartokinase III (EC 2.7.2.4) and 4-hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7) This is an operation to release the feedback inhibition for
  • Aspartokinase III is an enzyme that catalyzes the reaction that converts aspartic acid and adenosine triphosphate (ATP) into 4-phosphoaspartic acid and adenosine diphosphate. It is generally recognized that this enzyme is feedback-inhibited by L-lysine. A mutant enzyme that has been modified so as not to be subject to feedback inhibition changes its protein structure so that L-lysine does not bind, and it retains enzymatic activity even in the presence of lysine. It has been known. Aspartokinase III (lysC) derived from Eschericia coli will be described below as an example of an effective mutation, but the gene used in the present invention is not limited to this.
  • the mutant lysC that is not subject to feedback inhibition by L-lysine has an amino acid sequence in which the 352nd threonine residue is substituted with an isoleucine residue, and the 253rd threonine residue is substituted with an arginine residue. and the like, but are not limited to these.
  • 4-Hydroxy-tetrahydrodipicolinate synthase converts pyruvate and aspartate semialdehyde to (2S,4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinate and water is an enzyme that catalyzes It is generally recognized that this enzyme is feedback-inhibited by L-lysine.
  • a mutant enzyme that has been modified so that it is not subject to feedback inhibition changes its protein structure, does not bind L-lysine, and retains enzymatic activity even in the presence of lysine. are known.
  • dapA 4-hydroxy-tetrahydrodipicolinate synthase derived from Eschericia coli
  • the gene used in the present invention is not limited to this.
  • Mutant dapA that is not subject to feedback inhibition by L-lysine has an amino acid sequence in which the 81st alanine residue is replaced with a valine residue, and the 84th glutamic acid residue is replaced with a threonine residue. and those in which the histidine residue at position 118 is replaced with an arginine residue or a tyrosine residue, but are not limited to these.
  • the transformant or genome editor obtained as described above is cultured and maintained under conditions suitable for the growth and/or maintenance of the transformant for diamine production.
  • transformation with a vector having an expression cassette of an exogenous L-lysine decarboxylase gene each expression cassette may be placed on a separate vector or on the same vector.
  • the transformed transformant or the genome editor in which the expression cassette of the exogenous L-lysine decarboxylase gene is integrated into the genome sequence of the host microorganism is used for 1,5-pentanediamine production by growing the transformant and /or cultured and maintained under conditions suitable for maintenance.
  • Suitable medium composition, culture conditions and culture time for transformants derived from various host microbial cells can be easily determined by those skilled in the art.
  • the medium may be a natural, semi-synthetic, or synthetic medium containing one or more carbon sources, nitrogen sources, inorganic salts, vitamins, and optionally trace elements or trace components such as vitamins.
  • the medium used must adequately meet the nutritional requirements of the transformant to be cultured.
  • Carbon sources include D-glucose, sucrose, lactose, fructose, maltose, oligosaccharides, polysaccharides, starch, cellulose, rice bran, blackstrap molasses, fats and oils (such as soybean oil, sunflower oil, peanut oil, coconut oil, etc.), fatty acids ( palmitic acid, linoleic acid, linolenic acid, etc.), alcohols (eg, glycerol, ethanol, etc.), organic acids (eg, acetic acid, lactic acid, succinic acid, etc.). It can also be a biomass containing D-glucose. Suitable biomass includes corn hydrolyzate and cellulose hydrolyzate. Still other carbon sources include sugars, carbon dioxide, syngas, methanol, amino acids, and the like. These carbon sources can be used individually or as a mixture.
  • the product diamine can be obtained by measuring biobased carbon content based on Carbon-14 (radiocarbon) analysis specified in ISO 16620-2 or ASTM D6866, such as petroleum, natural gas, It can be clearly distinguished from synthetic raw materials derived from coal and the like.
  • Carbon-14 radiocarbon
  • Nitrogen sources include nitrogen-containing organic compounds (e.g. peptones, yeast extracts, meat extracts, malt extracts, corn steep liquor, soy flour and urea) or inorganic compounds (e.g. ammonium sulfate, ammonium chloride, phosphate ammonium, ammonium carbonate, sodium nitrate, ammonium nitrate, etc.). These nitrogen sources can be used individually or as a mixture.
  • organic compounds e.g. peptones, yeast extracts, meat extracts, malt extracts, corn steep liquor, soy flour and urea
  • inorganic compounds e.g. ammonium sulfate, ammonium chloride, phosphate ammonium, ammonium carbonate, sodium nitrate, ammonium nitrate, etc.
  • the medium may also contain a corresponding antibiotic if the transformant expresses useful additional traits, for example, has a marker for resistance to an antibiotic. This reduces the risk of contamination by germs during fermentation.
  • Antibiotics include, but are not limited to ampicillin, kanamycin, chloramphenicol, tetracycline, erythromycin, streptomycin, spectinomycin, and the like.
  • the host microorganism cannot assimilate the above carbon sources such as cellulose and polysaccharides, diamine production using these carbon sources by applying known genetic engineering techniques such as introducing exogenous genes into the host microorganism.
  • Exogenous genes include, for example, cellulase genes and amylase genes.
  • Cultivation may be batch or continuous. Moreover, in any case, it may be possible to replenish the additional carbon source or the like at an appropriate point in the culture. Furthermore, the culture should be continued while maintaining suitable temperature, oxygen concentration, pH and the like. Suitable culture temperatures for transformants derived from common microbial host cells are usually in the range of 15°C to 50°C, preferably 25°C to 37°C. If the host microorganism is aerobic, shaking (flask culture, etc.) and agitation/aeration (jar fermenter culture, etc.) should be performed to ensure an appropriate oxygen concentration during fermentation. Those culture conditions can be easily set by those skilled in the art.
  • a method for producing a diamine includes, for example, the following steps.
  • the method for producing diamine includes a culturing step of culturing the recombinant microorganism according to the above-described embodiment. By culturing in this step, a culture solution containing the cells is obtained. In the culturing step, the recombinant microorganism may be cultured in a culture solution containing an inorganic salt.
  • Inorganic salts are hydrochlorides, sulfates, phosphates, carbonates, hydrofluorides, etc. of metal elements, such as sodium chloride, lithium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, and sodium carbonate. Among them, sodium sulfate and sodium carbonate are preferred.
  • inorganic salt when added to an aqueous solution, its strong hydration force fixes water molecules as water of hydration, thus reducing the amount of water molecules required for hydration of diamines. It is thought that phase separation occurs.
  • the Hoffmeister series is an index indicating the strength of salting-out of inorganic salts, and salts composed of a combination of anions and cations (especially metal ions) that strongly cause salting-out, which are shown in the Hoffmeister series, are preferred.
  • Diamines are produced by culturing in a culture solution containing inorganic salts, but in the presence of a high concentration of inorganic salts, phase separation of the diamine aqueous solution occurs from the culture solution. Therefore, it is considered that the inclusion of an inorganic salt in the culture solution facilitates the separation of the diamine from the culture solution.
  • the recombinant composition is cultured in a culture medium containing, for example, 5% by mass or more of inorganic salt, preferably 10% by mass or more of inorganic salt.
  • the recombinant composition is cultured in a medium containing, for example, 5-20%, preferably 10-20% by weight of inorganic salts.
  • the phase containing inorganic salts can be reused as a culture solution after being separated from the culture solution by the separation process described below. Wastewater with a high concentration of salt is costly to treat, but recycling can reduce costs. It is also possible to reduce the load in the purification process, especially the load of dehydration and concentration due to phase separation. For example, a prior art has been proposed in which a strong base is added for diamine separation. is higher, and an additional solvent extraction step is required. As described above, by adding an inorganic salt to the culture solution, it is possible to reduce the load of dehydration and concentration in the purification process, and an additional step for solvent extraction becomes unnecessary.
  • Reaction step is a step of contacting a diamine precursor with a recombinant microorganism to produce the desired diamine from the diamine precursor. Contact with the diamine precursor may, for example, take place during or after the culturing step.
  • the diamine is 1,5-pentamethylenediamine
  • contacting the recombinant microorganism with lysine causes the lysine to be decarboxylated by lysine decarboxylase produced by the recombinant microorganism to produce 1,5-pentamethylenediamine.
  • a diamine is formed.
  • the culture solution and/or the bacterial cells obtained in the culture step are brought into contact with an aqueous solution containing 5% by mass or more of an inorganic salt and lysine to obtain 1,5-pentanediamine.
  • Obtain a reaction solution containing For example, in this step, the culture solution containing the cells obtained in the culture step, and/or the cells obtained by removing the supernatant from the culture solution obtained in the culture step by centrifugation or the like, is mixed with an inorganic salt and lysine. is brought into contact with an aqueous solution containing and to obtain a reaction liquid.
  • the culture step and the reaction step may be performed in the same step.
  • an aqueous solution containing an inorganic salt and lysine may be added to the culture medium for culturing the recombinant microorganism.
  • a bacterium that produces lysine by fermentation and the recombinant microorganism according to the present invention may be co-cultivated. By cocultivating them, the lysine produced by the fungus can be efficiently converted to 1,5-pentamethylenediamine by the lysine decarboxylase produced by the recombinant composition of the present invention.
  • the inorganic salt may be present in advance in the culture medium. That is, as explained in relation to the culture step, the recombinant microorganism according to the present invention may be cultured in a culture solution containing inorganic salts.
  • the recombinant composition is cultured in a culture medium containing, for example, 5% by mass or more of inorganic salt, preferably 10% by mass or more of inorganic salt.
  • the recombinant composition is cultured in a medium containing, for example, 5-20% by weight, preferably 10-20% by weight of inorganic salts.
  • the concentration of the inorganic salt in the culture medium is 100 to 200 g/L, preferably 150 to 200 g/L, more preferably 160 to 200 g/L, and even more preferably 200 g/L. L is added.
  • the inorganic salt may be brought into contact with the culture solution and/or the cells obtained in the culture step as an aqueous solution together with lysine in the reaction step.
  • inorganic salts may be added during both the culturing step and the reaction step.
  • inorganic salts may be added in one or more of the steps in the process for producing diamines, such as those described below, in addition to the culturing and/or reaction steps.
  • the inorganic salt is sodium carbonate or sodium sulfate, more preferably sodium sulfate.
  • a phase containing a diamine and an aqueous phase containing an inorganic salt can be phase-separated in the separation step described below, facilitating separation of the diamine.
  • the recombinant microorganism according to the present invention is a microorganism having halophilic properties, the growth of the microorganism is not inhibited even when such a high concentration of inorganic salt is added.
  • the advantage is that the separation of the diamine-containing phase can be facilitated without interfering with the production, and the diamine can be easily isolated.
  • the production method may further include a removal step of removing the recombinant microorganism from the culture solution or reaction solution.
  • the step is performed, for example, by centrifugation and/or filtration after diamine is produced by the culture step or reaction step. Through this step, solids such as bacterial cells contained in the culture solution or reaction solution can be removed.
  • an ultrafiltration membrane at the time of filtration, it is possible to remove high-molecular weight compounds having an arbitrary molecular weight or higher, including polysaccharides and proteins.
  • concentration step In this step, the culture solution or reaction solution is concentrated.
  • the concentration step is performed, for example, by concentrating the culture supernatant using, for example, an evaporator after removing the recombinant microorganism by the removal step.
  • concentration step is performed, for example, by concentrating the culture supernatant using, for example, an evaporator after removing the recombinant microorganism by the removal step.
  • the diamine is in one or more forms selected from the group consisting of carbonate, bicarbonate, bisbicarbonate, carbamate, and biscarbamate produced by the culturing step and/or reaction step
  • the concentration step the salt form of the diamine is converted to the free base and carbon dioxide, which is separated.
  • the diamine is 1,5-pentamethylenediamine
  • 1 selected from the group consisting of carbonate, bicarbonate, bisbicarbonate, carbamate, and biscarbamate produced by the culture step 1,5-Pentamethylenediamine, in more than one form, is converted to the free base and carbon dioxide in the concentration step, and the carbon dioxide is separated.
  • pH adjustment step the pH of the culture solution or the reaction solution is adjusted to 12 or more.
  • the pH of the concentrated culture solution or reaction solution is adjusted to 12 or higher.
  • it is confirmed that the pH is 12 or higher due to the pH increase due to the separation of carbon dioxide in the concentration step.
  • a phase containing diamine is separated from the culture solution or reaction solution.
  • the separation step does not involve adding alkaline compounds.
  • the alkali compound as used herein refers to a compound whose aqueous solution exhibits basicity and has the effect of increasing the pH value by adding it, and is particularly an inorganic alkali compound. Hydroxides of metal elements and inorganic substances capable of accepting hydrogen ions fall under this category. Examples of inorganic alkaline compounds include potassium hydroxide, sodium hydroxide, calcium hydroxide, lithium hydroxide, ammonia, and water.
  • Phase separation may be performed by adding an organic solvent to the culture solution or reaction solution, or by bringing the culture solution or reaction solution into contact with an organic solvent.
  • the organic solvent is, for example, one or more selected from the group consisting of n-hexane, n-butanol, and 2-ethyl-1-hexanol.
  • the diamine is 1,5-pentamethylenediamine, it is preferred to use n-butanol, and when the diamine is HMDA, it is preferred to use 2-ethyl-1-hexanol.
  • phase separation of diamines is promoted by inorganic salts of high salt concentration (for example, sodium carbonate and sodium sulfate), but an organic solvent may be added to further promote phase separation.
  • an organic solvent By adding an organic solvent, the extraction of the diamine from the aqueous phase to the organic phase can be promoted to increase the rate of transfer to the organic phase, thereby further improving the yield.
  • a phase containing an inorganic salt for example, containing an inorganic salt and water
  • the recovered phase is reused as a culture solution.
  • the recombinant microorganism according to the present invention has halophilic properties, it can produce diamine without inhibiting its growth even in a culture solution containing inorganic salts.
  • the inclusion of the inorganic salt promotes phase separation, making it easier to separate the diamine.
  • treatment of water containing a high concentration of inorganic salts may be costly, but by reusing the water as a culture medium for microorganisms as described above, wastewater treatment costs can be reduced.
  • (h) Purification step In this step, the diamine obtained from the culture is purified.
  • Methods for purifying diamines, such as 1,5-pentanediamine, from cultures are known to those skilled in the art.
  • 1,5-pentanediamine is present in the culture supernatant or within the cells, but may be extracted from the cultured cells if necessary.
  • the culture may be centrifuged to separate the supernatant from the cells, and the cells may be disrupted with a surfactant, organic solvent, enzyme or the like using a homogenizer.
  • Methods for purifying the culture supernatant and, in some cases, the bacterial cell extract include deproteinization using protein precipitation by pH adjustment, etc., removal of impurities by adsorption using activated carbon, and ionic purification using ion exchange resins. After removing the substance by adsorption, it is purified by extraction using a known solvent, distillation, or the like. Of course, it goes without saying that some steps may be omitted or additional purification steps such as chromatography may be performed depending on the purity desired for the product.
  • Yet another embodiment of the present invention also relates to a method of purifying diamines from cultures obtained using the aforementioned recombinant compositions.
  • the purification method may include, singly or in combination, each of the steps described above for the diamine production method.
  • the growth of Escherichia coli and coryneform bacteria used in conventional methods is significantly inhibited in an environment of pH 9 or higher.
  • the pH of the solution rises, it is necessary to control the pH within a range that does not inhibit the growth of microorganisms by appropriately adding an acid solution.
  • the recombinant microorganism according to the present invention has alkalophilicity that allows it to grow even in an alkaline environment, by using the microorganism for diamine production, there is no need to adjust the pH by adding an acid solution as in the prior art. , the complication of the diamine production process can be avoided.
  • the recombinant microorganism according to the present invention is halophilic, it can grow even in a salt-containing culture medium. Therefore, the waste liquid containing the salt added in the separation step can be reused for culturing the microorganism, and the waste water treatment cost can be reduced.
  • adding an inorganic salt to the culture solution can reduce the load of dehydration concentration in the purification process, and solvent extraction An additional process for is also unnecessary.
  • Invention B Halophilic and/or alkalophilic recombinant microorganism that suppresses N-acetyldiamine production
  • the genetically modified microorganism of the present invention is a halophilic and/or alkalophilic host microorganism with diamine-producing ability that has undergone one or more genetic alterations to suppress N-acetylase.
  • alkalophilic microorganisms that have alkalophilic properties that allow them to grow in an alkaline environment with a pH of 9 or higher, halophilic properties that allow them to grow in an environment with a NaCl concentration of 0.2M or higher, or those that have both of these properties. exist.
  • alkalophilic microorganisms that can be used as host microorganisms are a kind of extremophilic microorganisms that exhibit diverse distributions and are a general term for microorganisms that can grow even in an environment with a pH of 9 or higher.
  • alkalophilic microorganisms which can grow only in an environment of pH 9 or higher
  • facultative alkalophilic microorganisms which have an optimum growth pH of 9 or higher but can grow even below pH 9. Some of them can grow even in a strong alkaline environment with a pH of 12 or higher. All of these are alkalophilic microorganisms and can be host microorganisms in the present invention.
  • halophilic microorganisms that can be used as host microorganisms is a general term for microorganisms that can cope with high-concentration salt stress.
  • non-halophilic bacteria whose optimum growth salt concentration is 0-0.2M sodium chloride, and low halophilic bacteria whose optimum growth salt concentration is 0.2-0.5M.
  • Moderate halophiles whose optimum growth salt concentration is 0.5 to 2.5M, and high halophiles whose optimum growth salt concentration is 2.5 to 5.2M. All of these are halophilic microorganisms and can be host microorganisms in the present invention.
  • Halophilic, alkalophilic, or both microorganisms that can be used as host microorganisms of the present invention include various microorganisms, non-limiting examples of which include Bacillus, Halomonas, Halo Bacteroides, Salinibacter, Alkalinephilus, Clostridium, Anaeroblanca, and the like.
  • the host microorganism of the invention is preferably a bacterium of the genus Bacillus. Among microorganisms belonging to the genus Bacillus, Bacillus pseudofirmus, Bacillus halodurans, and Bacillus marmarensis are preferred, and Bacillus pseudofirmus is more preferred.
  • the recombinant microorganism according to the present invention is halophilic, it can grow even in a salt-containing culture medium. Therefore, the waste liquid containing the salt added in the separation step of separating the diamine-containing phase from the culture solution or the mixed solution (reaction solution) can be reused for culturing the microorganisms according to the present invention, thus reducing the wastewater treatment cost. can be reduced.
  • N-acetyldiamine production can be suppressed in the enzymatic conversion process or culture process.
  • the target diamine compound can be efficiently produced by suppressing the production of by-products.
  • N-acetylating enzyme refers to an enzyme that N-acetylates a diamine compound to produce an N-acetyldiamine compound.
  • N-acetyltransferase is synonymous with the terms “N-acetyltransferase” and “N-acetyltransferase” and these terms are used interchangeably herein.
  • N-acetyltransferase is an enzyme that catalyzes a reaction that acetylates the N-terminus of an amino acid. In the diamine production pathway, as a side reaction, it catalyzes a reaction that N-acetylates diamine to produce N-acetyldiamine. do.
  • a halophilic and/or alkalophilic host microorganism is subjected to one or more genetic modifications that inhibit N-acetyltransferase. , containing one or more genetic modifications that inhibit N-acetyltransferase.
  • the genetic modification is - modification that suppresses the expression of the endogenous gene encoding the N-acetyltransferase, or - A modification that reduces the activity of the N-acetyltransferase.
  • Inhibition of N-acetyltransferase in microorganisms can be achieved by, for example, using analytical methods such as ion chromatography, - no N-acetyl form is detected in the culture supernatant of the microorganism; It can be confirmed by at least one of: - no decrease in cadaverine as a substrate and - no detection of the N-acetyl form as a product during measurement of enzymatic activity using a cell lysate.
  • the recombinant microorganism of the present invention has suppressed or eliminated N-acetyldiamine compound-producing ability compared to the non-mutant strain containing no genetic modification.
  • One or more genetic modifications that inhibit N-acetyltransferase are modifications to inhibit the N-acetyldiamine biosynthetic pathway. Such genetic modifications are for example: - deleting part or all of the endogenous gene encoding the N-acetyltransferase in the host microorganism from the genome sequence of the host microorganism; -Introducing a mutation in the gene sequence of the N-acetyltransferase in the host microorganism that results in a loss of enzymatic function; - It is carried out by one or more of the methods of introducing mutations such as substitutions, insertions and deletions into the promoter site and/or the RBS site of the N-acetyltransferase.
  • Modifications to inhibit the N-acetyldiamine biosynthetic pathway include, for example: - deleting part or all of the endogenous gene encoding the N-acetyltransferase in the host microorganism from the genome sequence of the host microorganism; -Introducing a mutation in the gene sequence of the N-acetyltransferase in the host microorganism that results in a loss of enzymatic function; - It is carried out by one or more of the methods of introducing mutations such as substitutions, insertions and deletions into the promoter site and/or the RBS site of the N-acetyltransferase.
  • the production of N-acetyltransferase is suppressed in the recombinant microorganisms obtained by the above operations. That is, the recombinant microorganism according to the present invention has been modified by one or more genetic manipulations so as to suppress the production of N-acetyltransferase. Due to such modification, the recombinant microorganism of the present invention preferably has suppressed or eliminated N-acetyldiamine compound-producing ability compared to the non-mutant strain containing no genetic modification. is doing.
  • the genetic manipulation may be, for example, one or more genetic manipulations selected from the group consisting of (A), (B), (C) and (D) below.
  • A an operation of deleting an endogenous gene encoding the N-acetyltransferase from the host microorganism;
  • B reducing the copy number of the endogenous gene for said N-acetyltransferase in said host microorganism;
  • C an operation of introducing a mutation into the expression control region of the endogenous gene of the N-acetyltransferase in the host microorganism; and
  • D an expression control region of the endogenous gene of the N-acetyltransferase in the host microorganism. is replaced with a low-expressible exogenous regulatory region.
  • genetic modification for example, techniques for introducing any foreign gene sequence into the genome sequence into the cells of the host microorganism and techniques for removing unnecessary gene sequences from the genome sequence can be used.
  • the process of culturing the host microorganism into which the vector described above has been introduced it is possible to obtain a genetically modified strain in which the gene sequence of the chromosome has been modified by homologous recombination due to changes in temperature and medium composition.
  • the culture temperature is 37 to 43° C. or higher and that the medium contains 1 mM or higher 4-chlorophenylalanine.
  • Techniques that can be applied to introduce the vectors described above into host microorganisms include conventional cloning and transfection methods such as conjugative transfer, co-precipitation, protoplast fusion, electroporation, retroviral transfection, and the like. be. Examples thereof can be found in Gene Cloning and DNA Analysis, T.W. A. Brown, 2016, or Molecular Cloning: A Laboratory Manual (Fourth Edition): Sambrook et al., 2012.
  • a typical N-acetyltransferase gene is yjbC of Bacillus pseudofirmus.
  • the amino acid sequence of the Bacillus pseudofilamus yjbC enzyme is shown in SEQ ID NO: 23, and the nucleotide sequence of the Bacillus pseudofilamus yjbC gene (BpOF4_01925 (GenBank: ADC48452)) is shown in SEQ ID NO: 24 (FIGS. 3 and 4).
  • the N-acetyltransferase is (A-1) consisting of the amino acid sequence shown in SEQ ID NO: 23, (A-2) 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the amino acid sequence shown in SEQ ID NO: 23 or (A-3) 1 to the amino acid sequence shown in SEQ ID NO: 23. consisting of an amino acid sequence in which ⁇ 10, 1-7, 1-5, or 1-3 amino acids have been deleted, substituted, inserted and/or added; It has enzymatic activity that produces diamine compounds.
  • the N-acetyltransferase is (A-1) consisting of the amino acid sequence shown in SEQ ID NO: 23, (A-2) consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and having an enzymatic activity to N-acetylate a diamine compound to produce an N-acetyldiamine compound; or (A-3) consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and/or added to the amino acid sequence shown in SEQ ID NO: 23, and N-acetylating the diamine compound to obtain N - have enzymatic activity to produce acetyldiamine compounds;
  • the N-acetyltransferase consists of (A-1) the amino acid sequence shown in SEQ ID NO:23.
  • the N-acetyltransferase is (B-1) DNA consisting of the base sequence shown in SEQ ID NO: 24, (B-2) an enzyme that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 24 and N-acetylates a diamine compound to produce an N-acetyldiamine compound DNA encoding a protein having activity; (B-3) 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the base sequence shown in SEQ ID NO: 24 DNA encoding a protein consisting of a nucleotide sequence having sequence identity and having an enzymatic activity to N-acetylate a diamine compound to produce an N-acetyldiamine compound; (B-4) deletion of 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids from the amino
  • the N-acetyltransferase is (B-1) DNA consisting of the base sequence shown in SEQ ID NO: 24, (B-2) an enzyme that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 24 and N-acetylates a diamine compound to produce an N-acetyldiamine compound DNA encoding a protein having activity; (B-3) Consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 24, and having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound DNA that encodes a protein, (B-4) Encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and/or added to the amino acid sequence of the protein encoded by the nucleotide sequence shown in SEQ
  • stringent conditions are, for example, conditions of about “1 ⁇ SSC, 0.1% SDS, 60° C.”, and more stringent conditions are “0.1 ⁇ SSC, 0.1% SDS, 60° C. and a more severe condition is about "0.1 x SSC, 0.1% SDS, 68°C”.
  • the N-acetyltransferase is encoded by (B-1) DNA consisting of the nucleotide sequence shown in SEQ ID NO:24.
  • sequence identity As used herein, the percentage of "sequence identity" of a comparative amino acid sequence relative to a reference amino acid sequence is expressed by aligning the sequences so that the identity between these two sequences is maximized, and if necessary Defined as the percentage of amino acid residues in a comparison sequence that are identical to amino acid residues in a reference sequence when gaps are introduced in one or both of the two sequences. At this time, conservative substitutions are not considered part of sequence identity. Sequence identity can be determined by using publicly available computer software, for example, using an alignment search tool such as BLAST (registered trademark) (Basic Local Alignment Search Tool). can decide. Those skilled in the art can determine appropriate parameters for maximal alignment of the comparison sequences in the alignment. "Sequence identity" of nucleotide sequences can also be determined by similar methods.
  • the genetically modified microorganism obtained as described above is cultured and maintained under conditions suitable for its growth and/or maintenance for diamine production.
  • Appropriate medium compositions, culture conditions and culture times for transformants derived from various host microbial cells are selected by those skilled in the art.
  • the second aspect of the present invention relates to a method for producing a diamine compound, which includes culturing the aforementioned recombinant microorganism.
  • the production method includes a culturing step of culturing the aforementioned recombinant microorganism to obtain a culture of the recombinant microorganism and/or an extract of the culture.
  • the medium used in the culture process is as explained in Invention A above.
  • the product diamine can be clearly distinguished from synthetic raw materials by the measurement method described in Invention A.
  • the host microorganism if the host microorganism cannot assimilate carbon sources such as cellulose and polysaccharides, the host microorganism is adapted to diamine production using the carbon source by applying the genetic engineering technique described in Invention A. be able to.
  • the production method according to the present invention preferably further includes a mixing step of mixing the culture and/or the culture extract with a substrate compound to obtain a mixed solution.
  • a diamine compound, which is the target compound, is produced in the culture and/or mixed solution as a result of the reaction. Therefore, in a more preferred embodiment, the production method according to the present invention further includes a recovery step of recovering the diamine compound from the culture and/or the mixed solution.
  • the manufacturing method according to the present invention may include one or more selected from the steps described in Invention A.
  • the present invention provides a halophilic and/or alkalophilic recombinant microorganism capable of producing diamine, which is an N-acetyltransferase that N-acetylates a diamine compound to produce an N-acetyldiamine compound.
  • the production of N-acetyldiamine compounds, which are by-products can be suppressed.
  • the recombinant microorganism of the present invention has diamine-producing ability and can produce diamine while suppressing the production of by-products. Therefore, the target compound diamine can be efficiently obtained.
  • the recombinant microorganism according to the present invention is also expected to be applied to the production of diamine compounds on an industrial scale.
  • the recombinant microorganism according to the present invention is alkalophilic, by using the microorganism for diamine production, there is no need to adjust the pH by adding an acid solution, and it is possible to avoid complication of the diamine production process. is.
  • the recombinant microorganism according to the present invention is halophilic, it can grow even in a culture solution containing salt, and is added in the separation step of separating a phase containing diamine from the culture solution or mixed solution (reaction solution). Since the waste liquid containing the salt can be reused for culturing the microorganism, the waste water treatment cost can be reduced.
  • Example A1 Construction of cadA gene expression plasmid for Bacillus pseudofilamus and acquisition of transformant (Example A1-a) Cloning of promoter region Bacillus pseudofilamus OF4 strain (JCM17055 strain, this strain is (provided by RIKEN BRC through the Ministry of National Resources Project) was shake-cultured in 181 medium (2 ml) at 37°C. After the culture was completed, the cells were recovered from the culture medium, and genomic DNA was extracted using Nucleo Spin Tissue (product name, MACHEREY-NAGEL).
  • the promoter region (SEQ ID NO: 5) of the rpoD gene of Bacillus pseudofilamus was PCR-amplified (fragment 1) with a primer set having the sequences shown in SEQ ID NOS: 6 and 7.
  • the reaction conditions were 98° C. (10 sec), 55° C. (5 sec), 72° C. (30 sec), and 30 cycles.
  • Plasmid pAL351 (as NITE P-02918, National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary Center (NPMD) (Address: 2-5-8 Kazusa Kamatari, Chiba Prefecture, Room 122) in March 2019 18, and was subsequently transferred to an international deposit under the Budapest Treaty on July 28, 2020, with accession number NITE BP-02918.), SEQ ID NO: 8 and SEQ ID NO: 9.
  • PCR amplification was performed using the primer set shown in (Fragment 2). The reaction conditions were 98° C. (10 sec), 55° C. (5 sec), 72° C. (30 sec), and 30 cycles. With fragment 2 on the vector side, fragment 1 was ligated as an insert to construct pALP01.
  • Example A1-b Cloning of cadA gene Escherichia coli strain W3110 (NBRC12713) was shake-cultured in LB medium (2 mL) at 37°C. After completion of the culture, the cells were recovered from the culture medium, and genomic DNA was extracted using Nucleo Spin Tissue. Using the extracted genomic DNA as a template, PCR amplification was performed with a primer set having the sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11. The reaction conditions were 98° C. (10 sec), 55° C. (5 sec), 72° C. (30 sec), and 30 cycles. The amplified fragment was ligated downstream of the promoter sequence of pALP01 to construct pAL328.
  • Example A1-c Acquisition of transformant pAL328 constructed in Example A1-b, - Bacillus pseudofilamus OF4 strain (JCM17055 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project), - Bacillus halodurans C-125 strain (JCM9153 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project), - Bacillus marmaensis GMBE72 strain (JCM15719 strain, this strain was provided by RIKEN BRC via the Ministry of Education, Culture, Sports, Science and Technology National Resource Project) , respectively, ⁇ AKRM-1 strain, ⁇ AKRM-2 strain, ⁇ The AKRM-3 strain was acquired.
  • the plasmid pALP01 which does not contain the cadA gene, - Bacillus pseudofilamus OF4 strain (JCM17055 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project), - Bacillus halodurans C-125 strain (JCM9153 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project), - Bacillus marmaensis GMBE72 strain (JCM15719 strain, this strain was provided by RIKEN BRC via the Ministry of Education, Culture, Sports, Science and Technology National Resource Project) , respectively, ⁇ AKRM-4 strain, ⁇ AKRM-5 strain, ⁇ The AKRM-6 strain was acquired.
  • Bacillus pseudofilamus OF4 strain a transformant with a cadA gene expression cassette on the chromosome was also obtained.
  • This strain, Bacillus pseudofilamus AKAL-001 has been designated as NITE P-02920, National Institute of Technology and Evaluation, National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary Center (NPMD) (Address: 2-5 Kazusa Kamatari, Kisarazu City, Chiba Prefecture). -8 Room 122) on March 18, 2019, and then on July 28, 2020, it was transferred to an international deposit under the Budapest Treaty and was given accession number NITE BP-02920.
  • Example A2 Cadaverine production by constructed strain (flask culture) Each transformant obtained was cultured on a 181 medium plate at 37° C. for 2 days to form colonies. Put 2 mL of 181 medium in a 14 mL test tube, inoculate a colony from the plate with a platinum loop, culture at 37 ° C. and 180 rpm, culture until sufficient turbidity is obtained, and use this for the main culture It was used as a pre-culture solution.
  • BS medium composition shown in Table A-2
  • a 150 mL Erlenmeyer flask 30 mL was placed in a 150 mL Erlenmeyer flask, 0.3 mL of pre-culture solution was added, and main culture (cadaverine production test) was performed.
  • the culture conditions were 37° C. and 180 rpm.
  • 10 mg/L of chloramphenicol was added to the medium for both preculture and main culture.
  • the above culture solution was centrifuged at 10,000 g for 3 minutes, the supernatant was collected, and the cadaverine concentration of the culture supernatant was measured.
  • CG-19 guard column
  • CS-19 analytical column
  • ion chromatography analysis detector: electrical conductivity, Column temperature: 30°C, flow rate: 0.35 mL/min, mobile phase: 8 mM methanesulfonic acid aqueous solution ⁇ 70 mM methanesulfonic acid aqueous gradient
  • AKRM-1 strain the transformants of the present invention
  • AKRM-2 strain the transformants of the present invention
  • AKRM-3 strain the transformants of the present invention
  • AKAL-001 strain the transformants of the present invention
  • control strains AKRM-4 strain, AKRM-5 strain, AKRM-6 strain
  • Table A-3 The results of comparison with are shown in Table A-3.
  • the transformants of the present invention AKRM-1 strain, AKRM-2 strain, AKRM-3 strain, AKAL-001 strain
  • AKRM-1 strain AKRM-1 strain
  • AKRM-2 strain AKRM-2 strain
  • AKRM-3 strain AKAL-001 strain
  • AKAL-001 strain produced more cadaverine with the passage of culture time.
  • Example A3 Cadaverine production by constructed strain (fermenter culture) AKRM-1 strain, AKRM-4 strain, and AKAL-001 strain were cultured on a 181 medium plate at 37° C. for 2 days to form colonies. Put 100 mL of 181 medium in a 500 mL Erlenmeyer flask, inoculate a colony from the plate with a platinum loop, culture at 37 ° C. and 180 rpm until sufficient turbidity is obtained. It was used as a pre-culture solution.
  • a BS jar medium containing 20 g/L glucose (shown in Table A-4) was placed in a 10 L jar culture apparatus (model name: MDL-6C, manufactured by Marubishi Bioengineering Co., Ltd.), and 100 mL of the preculture solution was added.
  • a main culture (cadaverine production test) was performed.
  • 10 mg/L of chloramphenicol was added to the medium for both preculture and main culture.
  • the culture conditions were culture temperature: 37° C., culture pH: 7.5, alkali addition: 28% ammonia water, stirring speed: 700 rpm, aeration speed: 0.1 vvm.
  • a feed medium (composition shown in Table A-5) was sequentially added during the culture so that the glucose concentration in the medium was 0 to 5 g/L. Sampling was performed over time during the culture, and the cadaverine concentration in the culture supernatant was quantified.
  • the results are shown in Table A-6.
  • the AKRM-4 strain does not produce cadaverine
  • the AKRM-1 strain that expresses the cadA gene on the plasmid does not increase the concentration of cadaverine
  • the AKAL-001 strain that expresses the cadA gene on the chromosome decreases the cadaverine concentration with the passage of culture time. Cadaverine concentration increased accordingly.
  • Example A4 Cadaverine production with increasing sodium sulfate concentration (flask culture)
  • the AKAL-001 strain was cultured on a 181 medium plate at 37° C. for 2 days to form colonies. Put 2 mL of 181 medium in a 14 mL test tube, inoculate a colony from the plate with a platinum loop, culture at 37 ° C. and 180 rpm, culture until sufficient turbidity is obtained, and use this for the main culture It was used as a pre-culture solution.
  • the above culture solution was centrifuged at 10,000 g for 3 minutes, the supernatant was collected, and the cadaverine concentration of the culture supernatant was measured by the method described in Example A2, and the results are shown in Table A-7.
  • the AKAL-001 strain produced cadaverine even in the presence of sodium sulfate at a high concentration of 100 g/L, and showed an increase in cadaverine concentration with the passage of culture time.
  • Example A5 Cadaverine (1,5-diaminopentane, manufactured by Fuji Film Wako Chemical) and sodium chloride were added to a simulated liquid for confirming phase separation when salt compounds were added at various concentrations (the composition is shown in Table A-8). , sodium sulfate, and sodium carbonate at various concentrations to prepare 5 mL of simulated solutions. After mixing with a vortex mixer, the mixture was allowed to stand at room temperature for 10 minutes, and the state of phase separation and the amounts of the aqueous phase and the cadaverine phase were confirmed. Also, the concentration of cadaverine contained in the aqueous phase was measured, the amount of cadaverine remaining in the aqueous phase was calculated, and the transfer rate from the aqueous phase to the cadaverine phase was calculated.
  • Table A-9 shows the final solution composition and separation results.
  • "x" indicates that phase separation does not occur
  • “ ⁇ ” indicates that the formed diamine phase is less than 10% of the total liquid volume (for example, if the total liquid volume is 5 mL, diamine phase is less than 0.5 mL)
  • " ⁇ " indicates that the diamine phase formed is 10% or more of the total liquid volume by visual confirmation.
  • This evaluation criterion for phase separation applies to the following tables as well. As a result of the test, it was confirmed that the diamine phase separation occurred under the condition that each inorganic salt was added at a high concentration.
  • Example A6 Sodium sulfate and cadaverine (1,5-diaminopentane, manufactured by Fujifilm Wako Chemical Co., Ltd.) were added to a simulated liquid for confirming phase separation of cadaverine and hexamethylenediamine (HMDA) (the composition is shown in Table A-8). ) or hexamethylenediamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at various concentrations to prepare 5 mL of simulated solutions. After mixing with a vortex mixer, the mixture was allowed to stand at room temperature for 10 minutes, and the state of phase separation and the amounts of the aqueous phase and the cadaverine phase were confirmed. Also, the concentration of cadaverine contained in the aqueous phase was measured, the amount of cadaverine remaining in the aqueous phase was calculated, and the transfer rate from the aqueous phase to the cadaverine phase was calculated.
  • HMDA hexamethylenediamine
  • Table A-10 shows the final solution composition and separation results. It was confirmed that both cadaverine and hexamethylenediamine (HMDA) undergo phase separation in the presence of high-concentration sodium sulfate.
  • HMDA hexamethylenediamine
  • Example A7 Solvent extractability (cadaverine) when adding sodium carbonate at each concentration
  • concentration of sodium carbonate and cadaverine (1,5-diaminopentane, manufactured by Fuji Film Wako Chemical) was added to the simulated solution for confirmation of separation (composition shown in Table A-8) to prepare 5 mL of simulated solution. did.
  • An equal amount (5 mL) of n-butanol was added to the simulated solution, mixed with a vortex mixer, and allowed to stand at room temperature for 10 minutes.
  • the concentration of cadaverine contained in the aqueous phase was measured, the amount of cadaverine remaining in the aqueous phase was calculated, and the migration rate from the aqueous phase to the solvent phase was calculated.
  • Table A-11 shows the final solution composition and separation results. It was confirmed that the extraction efficiency into the solvent improved as the concentration of sodium carbonate increased.
  • Example A8 Solvent extractability (hexamethylenediamine) when sodium sulfate is added at various concentrations Each concentration of sodium sulfate and hexamethylenediamine was added to a simulated liquid for confirmation of separation (composition shown in Table A-8) to prepare 5 mL of simulated liquid. An equal amount (5 mL) of hexane or 2-ethyl-1-hexanol was added to the simulated solution, mixed with a vortex mixer, and allowed to stand at room temperature for 10 minutes. The concentration of hexamethylenediamine contained in the solvent phase was measured, and the migration rate from the aqueous phase to the solvent phase was calculated.
  • Table A-12 shows the final solution composition and separation results. It was confirmed that the extraction efficiency into 2-ethyl-1-hexanol improved as the concentration of sodium sulfate increased, while the extraction efficiency into n-hexane was very low.
  • Example A9 Phase separation in diamine-added culture medium 30 mL of the culture supernatant of the flask culture described in Example A4 was filtered through a 0.22 ⁇ m filter, and cadaverine (1,5-diaminopentane, manufactured by Fujifilm Wako Chemical ) or hexamethylenediamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each concentration, and concentrated twice with an evaporator (yield: 15 mL). After mixing the concentrate with a vortex mixer, the mixture was allowed to stand at room temperature for 10 minutes, and the state of phase separation and the amounts of the aqueous phase and the cadaverine phase were confirmed.
  • cadaverine 1,5-diaminopentane, manufactured by Fujifilm Wako Chemical
  • hexamethylenediamine manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • the concentration of cadaverine contained in the aqueous phase was measured, the amount of cadaverine remaining in the aqueous phase was calculated, and the transfer rate from the aqueous phase to the cadaverine phase was calculated.
  • Table A-13 shows the finally prepared solution composition and separation results. It was confirmed that both cadaverine and hexamethylenediamine undergo phase separation when high concentrations of sodium sulfate are present in the culture medium.
  • Example A10 Culture Reusing the Aqueous Phase After Phase Separation 100 mL of the fermenter culture medium (cultured for 63 hours) of the AKAL-001 strain described in Example A3 was recovered and centrifuged to recover the supernatant. 100 mL of the culture supernatant was filtered through a 0.22 ⁇ m filter and concentrated twice with an evaporator (yield: 50 mL). Hexamethylenediamine (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was added to the concentrate to a final concentration of 150 g/L. After mixing with a vortex mixer, the formation of phase separation was confirmed after standing at room temperature for 10 minutes.
  • Hexamethylenediamine manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.
  • the aqueous phase was 20 mL and the concentration of hexamethylenediamine was 48.5 g/L.
  • This aqueous phase was collected with a pipette, and after adjusting the pH to 7.5 with sulfuric acid, 30 mL of distilled water was added to make up to 50 mL.
  • This solution and fresh BS medium (composition shown in Table A-14) were mixed at different ratios and prepared in a 150 mL Erlenmeyer flask.
  • Bacillus pseudofilamus OF4 strain (JCM17055 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project) was cultured on a 181 medium plate at 37 ° C. for 2 days to form colonies. formed. Put 2 mL of 181 medium in a 14 mL test tube, inoculate a colony from the plate with a platinum loop, culture at 37 ° C. and 180 rpm, culture until sufficient turbidity is obtained, and use this for the main culture It was used as a pre-culture solution.
  • the main culture was performed by adding 0.1 mL of the pre-culture solution to the medium prepared in the flask.
  • the culture conditions were 37° C. and 180 rpm. After 49 hours of culture, the culture solution was sampled and turbidity (OD600) was measured.
  • the concentrations of diamine and inorganic salts increase, and further improvement in separation efficiency can be expected.
  • the step of adjusting pH by adding an acid solution can be omitted.
  • the water containing high concentrations of salts generated when liberating the diamine can be reused as a medium, and the wastewater treatment cost can be reduced.
  • Example B1 Acquisition of gene-disrupted strains From the annotation information of Bacillus pseudofilamus OF4 strain published on the NCBI database, 39 genes encoding acetyltransferase were identified as candidates encoding N-acetyltransferase of diamine. Selected as a gene.
  • - BpOF4_11255 gene (GenBank: ADC50304), - BpOF4_16515 gene (GenBank: ADC51348), - BpOF4_18375 gene (GenBank: ADC51716), - BpOF4_16725 gene (GenBank: ADC51388), - BpOF4_18160-65 gene (GenBank: ADC51673-4), - BpOF4_18545 gene (GenBank: ADC51750), - BpOF4_19380 gene (GenBank: ADC51915), - BpOF4_00750 gene (GenBank: ADC48219), ⁇ BpOF4_01925 gene (GenBank: ADC48452) was generated by gene manipulation to the chromosome by homologous recombination. The primer sequences used are shown in Figures 5A and 5B.
  • Example B1-a Preparation of gene disruption plasmid Bacillus pseudofilamus OF4 strain (JCM17055 strain, this strain was provided by Riken BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project) in 181 medium (2 ml) Shaking culture was carried out at 37°C. After the culture was completed, the cells were recovered from the culture medium, and genomic DNA was extracted using Nucleo Spin Tissue (product name, MACHEREY-NAGEL). Using a primer set with "A” and "B” at the end of the primer name and a primer set with "C” and “D” at the end of the primer name (see FIG. 5), the homologous region was identified. Fragments 1 and 2 were obtained by PCR amplification. The reaction conditions were 98° C. (10 sec), 55° C. (5 sec), 72° C. (30 sec), 30 cycles.
  • Plasmid pAL351 (Deposited on March 18, 2019 at National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary Center (NPMD). Accession number: NITE BP-02918) is added to the end of the primer name. Fragment 3 was obtained by PCR amplification using the primer set labeled “E” and “F” (see FIG. 5). The reaction conditions were 98° C. (10 sec), 55° C. (5 sec), 72° C. (30 sec), 30 cycles. Fragment 3 was used as a vector, and fragments 1 and 2 were ligated as inserts to construct pAKNU01-09.
  • Example B1-b Acquisition of transformants pAKNU01 to 09 constructed in Example B1-a were transformed into Bacillus pseudofilamus AKAL-001 strain (National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary Center ( NPMD), accession number: NITE BP-02920), and strains AKALp-101 to AKALp-109 were obtained.
  • Bacillus pseudofilamus AKAL-001 strain National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary Center ( NPMD), accession number: NITE BP-02920
  • strains AKALp-101 to AKALp-109 were obtained.
  • Example B1-c Acquisition of Chromosome Insertion Strains AKALp-101 to AKALp-109 strains obtained in Example B1-b were cultured at 30° C. for 1 day in 181 medium containing 10 ⁇ g/mL chloramphenicol. After that, it was diluted 100-fold and applied to 181 medium containing 10 ⁇ g/mL of chloramphenicol, cultured at 43° C., and chromosomally inserted strains AKALp-201 strain to AKALp-209 strain in which the plasmid was homologously recombined into the chromosome. Acquired.
  • Example B1-d Acquisition of yjbC gene-disrupted strain
  • the strains AKALp-201 to AKALp-209 obtained in Example B1-c were cultured at 30°C for 1 day in 181 medium containing 10 ⁇ g/mL chloramphenicol. After that, it was diluted 100 times and applied to 181 medium containing 5 mM 4-chlorophenylalanine, cultured at 30 ° C., added to the plasmid from the chromosome, and the gene disruption strain AKDNC-001 strain to which each gene region was removed.
  • AKDNC-009 strain was obtained. Gene deletion was confirmed by PCR using a primer set with suffixes "G" and "H” (see FIG. 5). The reaction conditions were 98° C. (10 sec), 55° C. (5 sec), 72° C. (30 sec), 30 cycles.
  • Example B2 Evaluation of N-acetylcadaverine-producing ability in constructed strains (fermenter culture)
  • Strains AKAL-001 and AKDNC-001 to 009 were cultured on 181 medium plates at 37° C. for 1 day to form colonies. Put 2 mL of 181 medium in a 14 mL test tube, inoculate a colony from the plate with a platinum loop, culture at 37 ° C. and 180 rpm, culture until sufficient turbidity is obtained, and use this for the main culture It was used as a pre-culture solution.
  • BS jar medium containing 30 g/L glucose (shown in Table B-3) is placed in a 100 mL jar culture apparatus (model name: Bio Jr. 8, manufactured by Biot), 1 mL of preculture solution is added, and main culture is performed. was performed (cadaverine production test).
  • the culture conditions were culture temperature: 37° C., culture pH: 7.5, addition of alkali: 10% aqueous ammonia, stirring speed: 750 rpm, aeration speed: 0.1 vvm. Sampling was performed over time during the culture, and the cell turbidity in the culture medium and the diamine concentration in the culture supernatant were quantified. 6 ml of 50% glucose solution was added 24 hours after the initiation of culture.
  • the above culture medium was centrifuged at 10,000 g ⁇ 3 minutes, the supernatant was collected, and the cadaverine concentration and N-acetylcadaverine concentration of the culture supernatant were measured.
  • CG-19 guard column
  • CS-19 analytical column
  • mobile phase gradient of 8 mM methanesulfonic acid aqueous solution ⁇ 70 mM methanesulfonic acid aqueous solution
  • concentration of cadaverine and N-acetylcadaverine in the culture supernatant was adjusted. quantified.
  • Table B-4 shows the cell density (OD), cadaverine and N-acetylcadaverine concentrations (g/l) after 40 hours of culture.
  • Cadaverine and N-acetylcadaverine were detected in the culture supernatants of AKAL-043 and AKDNC-001-008 strains.
  • cadaverine was detected in the culture supernatant of the AKDNC-009 strain, but N-acetylcadaverine was not detected (Table B-4).
  • the AKDNC-009 strain had a 14% increase in cadaverine concentration compared to the AKAL-043 strain.
  • the strains in which the production of acetylcadaverine is 0.5 g/l or more lower than the non-mutant strain AKAL-001 produce more N-acetyldiamine compounds than the non-mutant strain. It is understood that the activity is lowered compared to the wild type.
  • the present invention can be used for industrial fermentation production of diamine. Since the present invention can also suppress the production of by-products and efficiently produce diamine, it is expected that the fermentation of the recombinant microorganism of the present invention can be used for industrial-scale production of diamine.

Abstract

[Problem] To provide: with respect to the production of a diamine, a recombinant microorganism and a method for manufacturing a diamine, that realize a simplified process and/or a reduction in effluent treatment costs; and a halophilic and/or alkalophilic recombinant microorganism in which N-acetyl diamine production is suppressed. [Solution] The present invention provides a halophilic and/or alkalophilic recombinant microorganism having a diamine producing ability, wherein the diamine is represented by the formula NH2CH2(CH2)nCH2NH2 (in the formula, n is an integer between 0 and 10), the halophilic and/or alkalophilic recombinant microorganism resulting from modification of a halophilic and/or alkalophilic host microorganism so as to impart a diamine-producing ability; and a halophilic and/or alkalophilic recombinant microorganism having an amine producing ability, the recombinant microorganism having one or more genetic modifications for suppressing an N-acetylation enzyme which N-acetylates a diamine compound and generates an N-acetyl diamine compound.

Description

ジアミン生産能を有する組換え微生物およびジアミンの製造方法Recombinant microorganism having diamine-producing ability and method for producing diamine
　本発明は、産業上有用な化合物であるジアミン生産能を有する、好塩性および／または好アルカリ性の組換え微生物、および、当該組換え微生物を用いてジアミンを製造する方法に関する。さらに本発明は、Ｎ－アセチルジアミン生産を抑制した好塩性および／または好アルカリ性の組換え微生物にも関する。 The present invention relates to a halophilic and/or alkalophilic recombinant microorganism capable of producing diamine, which is an industrially useful compound, and a method for producing diamine using the recombinant microorganism. The present invention also relates to halophilic and/or alkalophilic recombinant microorganisms with suppressed N-acetyldiamine production.
　近年の地球温暖化に伴う災害および気候変動をうけ、地球環境との共生および環境保全を目指した、サスティナブルなプロセスの開発が強く求められている。その中で、再生可能原料を利用することができ、かつ、生体反応を利用した物質生産工程であるバイオプロセスに大きな期待が寄せられている。これまでに、さまざまな化成品の発酵生産プロセスが開発されている。例えば、ポリウレタン原料、ポリエステル原料、可塑剤原料および医薬中間体等に利用されるポリオール類の発酵生産プロセスが提案されている。 In response to the recent disasters and climate change associated with global warming, there is a strong demand for the development of sustainable processes aimed at coexistence with the global environment and environmental conservation. Among them, great expectations are placed on bioprocesses, which are material production processes that make use of renewable raw materials and utilize biological reactions. So far, fermentative production processes for various chemical products have been developed. For example, a fermentative production process for polyols used for polyurethane raw materials, polyester raw materials, plasticizer raw materials, pharmaceutical intermediates, and the like has been proposed.
　ジアミンは、アジピン酸などのジカルボン酸と共に、ポリアミド（ＰＡ６６（６，６－ナイロン）など）の原料として工業的に重要な化合物である。 Diamine, along with dicarboxylic acids such as adipic acid, is an industrially important compound as a raw material for polyamide (PA66 (6,6-nylon), etc.).
　例えば、１，６－ヘキサメチレンジアミン（１，６－ジアミノヘキサンまたはヘキサメチレンジアミンとも呼ばれ、分子式Ｃ６Ｈ１２Ｎ２を有する化合物である。）は、ナイロン６６（ＰＡ６６）などの原料として、繊維および樹脂用途に幅広く利用され、世界的に需要が見込まれている。また、ヘキサメチレンジアミンは、イソシアネートを経由したウレタン原料、農薬、および医薬の中間体として利用されている。ヘキサメチレンジアミンは、ブタジエンのヒドロシアン化、アクリロニトリルの電解二量化、または、アジピン酸のニトリル化によってアジポニトリルを得て、さらにニッケルなどを触媒とした水素付加により合成される（非特許文献１）。本方法により、ヘキサメチレンジアミンは工業的に生産されているが、一旦、アジポニトリルの合成を行った後、水素付加反応を行う。 For example, 1,6-hexamethylenediamine (also called 1,6-diaminohexane or hexamethylenediamine, which is a compound having the molecular formula C 6 H 12 N 2 ) is used as a raw material for nylon 66 (PA66), etc. It is widely used for fiber and resin applications, and global demand is expected. Hexamethylenediamine is also used as an intermediate for urethane raw materials, agricultural chemicals, and pharmaceuticals via isocyanate. Hexamethylenediamine is synthesized by hydrocyanation of butadiene, electrolytic dimerization of acrylonitrile, or nitrilation of adipic acid to obtain adiponitrile, which is then hydrogenated using a catalyst such as nickel (Non-Patent Document 1). Although hexamethylenediamine is industrially produced by this method, once adiponitrile is synthesized, a hydrogenation reaction is carried out.
　微生物を用いたヘキサメチレンジアミンの製造法としては、細胞内のジカルボン酸、アミノカルボン酸およびジアルデヒドなどから、外来酵素、例えばカルボン酸脱炭酸酵素およびアミノトランスフェラーゼを組み合わせてジアミンを生産する方法が報告されている（特許文献１および特許文献２）。特許文献１では、ヘキサメチレンジアミン生産経路を有するように改変された宿主微生物において、欠失および／または破壊によって収率向上が予想される酵素遺伝子を、インシリコでの代謝シミュレーションを基に予想し、例示している。しかしながら、ヘキサメチレンジアミン生産経路中の中間体に由来する副生物およびその抑制方法についてはなんら言及していない。特許文献２は、６－ヒドロキシヘキサン酸を経由した酵素反応経路によるヘキサメチレンジアミンの生産方法を記載している。しかしながら、遺伝子組換えにより新規に構築されたヘキサメチレンジアミン生産経路中の中間体に由来する副生物の生成、およびその抑制方法については言及していない。 As a method for producing hexamethylenediamine using microorganisms, a method has been reported in which diamine is produced from intracellular dicarboxylic acids, aminocarboxylic acids, and dialdehydes by combining exogenous enzymes such as carboxylic acid decarboxylase and aminotransferase. (Patent Document 1 and Patent Document 2). In Patent Document 1, in a host microorganism modified to have a hexamethylenediamine production pathway, an enzyme gene whose yield is expected to be improved by deletion and/or disruption is predicted based on an in silico metabolic simulation, exemplified. However, no mention is made of by-products derived from intermediates in the hexamethylenediamine production pathway and methods of suppressing them. Patent Document 2 describes a method for producing hexamethylenediamine by an enzymatic reaction pathway via 6-hydroxyhexanoic acid. However, it does not refer to the generation of by-products derived from intermediates in the hexamethylenediamine production pathway newly constructed by genetic recombination, and a method for suppressing them.
　１，５－ペンタメチレンジアミン（１，５－ジアミノペンタンまたはカダベリンとも呼ばれる、分子式Ｃ５Ｈ１４Ｎ２を有する化合物である。）はナイロン（ＰＡ５６）の原料として、繊維、樹脂用途での需要が見込まれている。ＰＡ５６繊維は、ＰＡ６６と同等の強度と耐熱性を持ち、高吸湿性、高放湿性を示すことから、新たな市場展開が期待されている。また、１，５－ペンタメチレンジアミンはイソシアネートを経由したウレタン原料、農薬および医薬の中間体としても注目が集まりつつある。 1,5-Pentamethylenediamine (also called 1,5-diaminopentane or cadaverine, a compound with the molecular formula C 5 H 14 N 2 ) is in demand as a raw material for nylon (PA56) for fiber and resin applications. expected. PA56 fiber has the same strength and heat resistance as PA66, and exhibits high hygroscopicity and high hygroscopicity, so new market development is expected. 1,5-Pentamethylenediamine is also attracting attention as a raw material for urethane via isocyanate, an intermediate for agricultural chemicals and pharmaceuticals.
　１，５－ペンタメチレンジアミンの石化原料からの効率的な化学合成法はいまだ確立されていない一方、生合成においてはＬ－リジンの酵素的脱炭酸により容易に生成することが知られており、バイオマス由来の１，５－ペンタメチレンジアミンを原料とするＰＡ５６樹脂は、バイオプラスチックの一つとして、環境負荷の低減という観点から工業的に注目されている（非特許文献２）。 While an efficient chemical synthesis method from petrochemical raw materials of 1,5-pentamethylenediamine has not yet been established, it is known to be easily produced by enzymatic decarboxylation of L-lysine in biosynthesis. PA56 resin, which is made from biomass-derived 1,5-pentamethylenediamine, has been attracting industrial attention as one of bioplastics from the viewpoint of reducing environmental load (Non-Patent Document 2).
　また、再生可能原料からのジアミン類の発酵生産プロセスも提案されている。例えば、発酵生産可能なアミン化合物として、１，５－ペンタメチレンジアミンおよびヘキサメチレンジアミンが挙げられ、これらは、化成品の製造において、ポリマー製造のための原料として用いることができる（特許文献３～７および非特許文献３）。 A fermentative production process for diamines from renewable raw materials has also been proposed. For example, fermentation-producible amine compounds include 1,5-pentamethylenediamine and hexamethylenediamine, which can be used as raw materials for polymer production in the production of chemical products (Patent Documents 3 to 7 and Non-Patent Document 3).
　微生物による１，５－ペンタメチレンジアミンの製造法としては、リジン脱炭酸酵素を高発現させた大腸菌を培養し、前駆体であるリジンと当該酵素とを反応させ脱炭酸して１，５－ペンタメチレンジアミンを得る方法が知られている（特許文献８）。また、リジン脱炭酸酵素遺伝子の活性を増大させ、リジン生合成に重要な役割を果たす遺伝子の活性を低下させたコリネ型細菌を培養し、グルコースから１，５－ペンタメチレンジアミンを生産する方法も提案されている（特許文献９）。 As a method for producing 1,5-pentamethylenediamine using microorganisms, E. coli in which lysine decarboxylase is highly expressed is cultured, and the precursor lysine is reacted with the enzyme to decarboxylate 1,5-pentamethylenediamine. A method for obtaining methylenediamine is known (Patent Document 8). There is also a method of producing 1,5-pentamethylenediamine from glucose by culturing coryneform bacteria in which the activity of the lysine decarboxylase gene is increased and the activity of the gene that plays an important role in lysine biosynthesis is decreased. It has been proposed (Patent Document 9).
　一方、培養液からジアミンを分離精製する方法としては、例えば、水酸化ナトリウムなどのアルカリ溶液を加えて１，５－ペンタメチレンジアミンを遊離させた後、適切な溶媒を用いることにより抽出する方法が知られている（特許文献１０、１１および１２）。また、１，５－ペンタメチレンジアミン炭酸塩水溶液を熱分解し、粗１，５－ペンタメチレンジアミンと二酸化炭素とに分離した後、ジアミンを蒸留精製する方法も提案されている（特許文献１３）。さらに、微生物によるジアミン発酵生産において、外部から添加されるかまたは代謝によって発生する二酸化炭素により中和されたジアミン炭酸塩類および／またはジアミンカルバミン酸塩類から、ジアミンの遊離塩基と二酸化炭素とを分離（解離）した後、有機溶媒によってジアミンを抽出することが提案されている（特許文献１４）。 On the other hand, as a method for separating and purifying diamine from the culture medium, for example, an alkaline solution such as sodium hydroxide is added to liberate 1,5-pentamethylenediamine, and then an appropriate solvent is used for extraction. known (Patent Documents 10, 11 and 12). A method has also been proposed in which an aqueous solution of 1,5-pentamethylenediamine carbonate is thermally decomposed to separate crude 1,5-pentamethylenediamine and carbon dioxide, and then the diamine is purified by distillation (Patent Document 13). . Furthermore, in diamine fermentative production by microorganisms, diamine free bases and carbon dioxide are separated from diamine carbonates and/or diamine carbamates neutralized by carbon dioxide added from the outside or generated by metabolism ( Dissociation) and then extraction of the diamine with an organic solvent has been proposed (Patent Document 14).
　しかしながら、これまでに提案された上記の方法には、次のような不都合があった。まず、微生物によるジアミンの発酵生産に関して、例えば、リジンが１，５－ペンタメチレンジアミンに変換されると、培地中のｐＨが上昇するが、特許文献８および９に記載の方法で用いられる大腸菌およびコリネ型細菌は一般的にｐＨ７～８付近でのみ生育することができ、ｐＨ９以上の環境では生育が著しく阻害される。このため、このような微生物を触媒として利用する場合は、酸もしくはアルカリ溶液を適宜加えることでｐＨを微生物の生育を阻害しない範囲に制御する必要があり、製造プロセスが煩雑となる。 However, the above methods proposed so far have the following disadvantages. First, regarding fermentative production of diamine by microorganisms, for example, when lysine is converted to 1,5-pentamethylenediamine, the pH in the medium rises. Coryneform bacteria can generally grow only in the vicinity of pH 7-8, and their growth is markedly inhibited in an environment of pH 9 or higher. Therefore, when such microorganisms are used as a catalyst, it is necessary to control the pH within a range that does not inhibit the growth of microorganisms by appropriately adding an acid or alkaline solution, which complicates the production process.
　次に、ジアミンの精製工程に関しても、特許文献１１および１４に記載の方法では、抽出にクロロホルム、ヘキサン等の極性有機溶媒を用いるが、有機溶媒には有害性があるものが多く、その取扱いは好ましくない。また、抽出効率の低さが製造コストに大きく影響する。さらに、製造コスト低減のために、使用する有機溶媒を回収すると、製造プロセスが煩雑になる。また、特許文献１２に記載の方法では、晶析法によって精製を行っているが、晶析率は４０～４５％であり、高い収率を期待できない。 Next, with respect to the diamine purification process, the methods described in Patent Documents 11 and 14 use polar organic solvents such as chloroform and hexane for extraction, but many organic solvents are harmful and should be handled properly. I don't like it. In addition, the low extraction efficiency greatly affects the production cost. Furthermore, if the used organic solvent is recovered in order to reduce the production cost, the production process becomes complicated. In addition, in the method described in Patent Document 12, purification is performed by a crystallization method, but the crystallization rate is 40 to 45%, and a high yield cannot be expected.
　特許文献１０に記載の方法では、ジアミン遊離工程において大量の塩が副産物として発生する。例えば、硫酸を添加しながら中性を維持する培養を行って５０ｇ／Ｌのカダベリンを生産する場合、カダベリンのカウンターイオンである硫酸イオンの存在により最大１００ｇ／Ｌ程度の硫酸ナトリウムが副産物として発生する。この副産物を含有する廃液を処理するためには多大な費用がかかる可能性が高い。更に、特許文献１４に記載の方法では、無機塩類を含む、ジアミン溶媒抽出後の水相の取り扱いには言及していないが、この方法も廃液処理のために多大な費用がかかる可能性がある。 In the method described in Patent Document 10, a large amount of salt is generated as a by-product in the diamine liberation step. For example, when 50 g/L of cadaverine is produced by culturing to maintain neutrality while adding sulfuric acid, a maximum of about 100 g/L of sodium sulfate is generated as a by-product due to the presence of sulfate ions, which are counter ions of cadaverine. . It is likely to be very costly to dispose of the effluent containing this by-product. Furthermore, the method described in Patent Document 14 does not mention the handling of the aqueous phase after diamine solvent extraction, which contains inorganic salts, but this method may also require a great deal of cost for waste liquid treatment. .
　このように、既存のジアミン生産には、さらなる改良の余地があった。 Thus, there was room for further improvement in existing diamine production.
特開２０１５－１４６８１０号公報JP 2015-146810 A 特開２０１７－５４４８５４号公報JP 2017-544854 A 特開２０１２－１８８４０７号公報JP 2012-188407 A 特表２０１２－５２５８５６号公報Japanese Patent Publication No. 2012-525856 特表２０１６－５３８８７０号公報Japanese Patent Publication No. 2016-538870 特表２０１６－５０１０３１号公報Japanese Patent Publication No. 2016-501031 特表２０１７－５３３７３４号公報Japanese Patent Publication No. 2017-533734 特許第５５５３３９４号公報Japanese Patent No. 5553394 特許第５２１０２９５号公報Japanese Patent No. 5210295 特許第５６４６３４５号公報Japanese Patent No. 5646345 特許第４１９６６２０号公報Japanese Patent No. 4196620 特開２００５－６６５０号公報JP-A-2005-6650 特許第５９３０５９４号公報Japanese Patent No. 5930594 特表２０１８－５００９１１号公報Japanese Patent Publication No. 2018-500911
　上記の現状に鑑み、本発明の課題は、ジアミンの生産において、プロセスの簡略化及び排水処理コストの削減の少なくとも一方を実現可能とする組換え微生物およびジアミンの製造方法を提供することである。 In view of the above-mentioned current situation, an object of the present invention is to provide a recombinant microorganism and a diamine production method that can achieve at least one of process simplification and wastewater treatment cost reduction in the production of diamine.
　本発明の更なる課題は、Ｎ－アセチルジアミン生産が抑制された、好塩性および／または好アルカリ性の組換え微生物を提供することである。 A further object of the present invention is to provide a halophilic and/or alkalophilic recombinant microorganism in which N-acetyldiamine production is suppressed.
　上記の課題を解決するために、発明者らは鋭意検討を行った結果、好塩性及び好アルカリ性の少なくとも一方を有する宿主微生物を、ジアミン生産能を有するように改変させて得られる本発明の組換え微生物により、ジアミンの生産において、プロセスの簡略化及び排水処理コストの削減の少なくとも一方を実現できることを見出した。 In order to solve the above problems, the inventors conducted extensive studies and found that the host microorganism of the present invention, which has at least one of halophilicity and alkalophilicity, is modified to have diamine-producing ability. It has been found that recombinant microorganisms can achieve at least one of process simplification and wastewater treatment cost reduction in diamine production.
　すなわち本発明は以下を提供する：
［１］ジアミン生産能を有する、好塩性および／または好アルカリ性の組換え微生物であって、
　該ジアミンが、式：ＮＨ２ＣＨ２(ＣＨ２)ｎＣＨ２ＮＨ２（式中、ｎは０～１０の整数である）で表され、
　好塩性および／または好アルカリ性の宿主微生物に、ジアミン生産能を有するように改変が行われた、組換え微生物；
［２］前記式中におけるｎが３または４である、［１］に記載の組換え微生物；
［３］Ｌ－リジンデカルボキシラーゼ（ＥＣ　４．１．１．１８）の過剰生産を誘導するように、宿主微生物が１つ以上の遺伝子操作により改変された［２］に記載の組換え微生物；
［４］前記遺伝子操作が、下記（Ａ）、（Ｂ）、（Ｃ）、（Ｄ）及び（Ｅ）：
（Ａ）前記宿主微生物に、前記Ｌ－リジンデカルボキシラーゼをコードする外来性遺伝子を導入する操作、
（Ｂ）前記宿主微生物内の前記Ｌ－リジンデカルボキシラーゼの内因性遺伝子のコピー数を増加させる操作、
（Ｃ）前記宿主微生物内の前記Ｌ－リジンデカルボキシラーゼの内因性遺伝子の発現調整領域に変異を導入する操作、
（Ｄ）前記宿主微生物内の前記Ｌ－リジンデカルボキシラーゼの内因性遺伝子の発現調整領域を、高発現可能な外来調整領域で置換する操作、および
（Ｅ）前記宿主微生物内の前記Ｌ－リジンデカルボキシラーゼの内因性遺伝子の調整領域を欠失させる操作
から成る群より選択される１以上の操作である、［３］に記載の組換え微生物；
［５］配列番号２または配列番号４に示す塩基配列と、８５、９０、９２、９５、９８または９９％以上の相同性を有する塩基配列を含むか、あるいは
　配列番号１または３に示すアミノ酸配列をコードする塩基配列と８５、９０、９２、９５、９８または９９％以上の相同性を有する塩基配列を含む、［２］～［４］のいずれか１項に記載の組換え微生物；
［６］リジン生産能を向上させるための突然変異操作または遺伝子組換え操作によりさらに改変されている、［２］～［５］のいずれか１項に記載の組換え微生物；
［７］前記突然変異操作または前記遺伝子組換え操作が、アスパルトキナーゼＩＩＩ（ＥＣ　２．７．２．４）及び４－ヒドロキシ－テトラヒドロジピコリン酸シンターゼ（ＥＣ　４．３．３．７）の少なくとも一方に対するフィードバック阻害を解除する操作である、［６］に記載の組換え微生物；
［８］前記宿主微生物が、バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｅｕｄｏｆｉｒｍｕｓ）、バチルス・ハロデュランス（Ｂａｃｉｌｌｕｓ　ｈａｌｏｄｕｒａｎｓ）、及びバチルス・マーマエンシス（Ｂａｃｉｌｌｕｓ　ｍａｒｍａｒｅｎｓｉｓ）からなる群より選択される、［１］～［７］のいずれか１項に記載の組換え微生物；
［９］前記宿主微生物が、バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｅｕｄｏｆｉｒｍｕｓ）である［１］～［８］のいずれか１項に記載の組換え微生物；
［１０］［１］～［９］のいずれか１項に記載の組換え微生物を用いて、式：ＮＨ２ＣＨ２(ＣＨ２)ｎＣＨ２ＮＨ２（式中、ｎは０～１０の整数である）で表されるジアミンを製造する方法；
［１１］前記式中におけるｎが３または４である、［１０］に記載の製造方法；
［１２］前記組換え微生物を５質量％以上の無機塩を含有する培地で培養することによりジアミンを含む培養液を得る培養工程を含む、［１０］または［１１］に記載の製造方法；
［１３］前記組換え微生物を培養して、菌体を含む培養液を得る培養工程と、
　前記培養液および／または前記菌体を、５質量％以上の無機塩とリジンとを含有する水溶液と接触させて１，５－ペンタンジアミンを含む反応液を得る反応工程と
を含む、［１０］～［１２］のいずれか１項に記載の製造方法；
［１４］前記培養液または反応液から前記組換え微生物を除去する除去工程を含む、［１２］または［１３］に記載の製造方法；
［１５］前記培養液または反応液を濃縮する濃縮工程を含む、［１４］に記載の製造方法；
［１６］前記濃縮工程により前記培養液または反応液を濃縮した後に、濃縮した培養液または反応液のｐＨを１２以上に調整するｐＨ調整工程を含む、［１５］に記載の製造方法；
［１７］前記培養液または反応液から、ジアミンを含む相と、前記無機塩を含む水相とを相分離する分離工程を含む、［１２］～［１６］のいずれか１項に記載の製造方法；
［１８］前記分離工程において、前記培養液または反応液に有機溶媒を添加することにより、ジアミンおよび有機溶媒を含む相と水相とに相分離する、［１２］～［１７］のいずれか１項に記載の製造方法；
［１９］前記分離工程において、アルカリ化合物を添加しない、［１７］または［１８］に記載の製造方法；
［２０］前記無機塩が、炭酸ナトリウム及び／又は硫酸ナトリウムである、［１２］～［１９］のいずれか１項に記載の製造方法；
［２１］前記培養工程及び／又は反応工程により生産したジアミンが、炭酸塩、重炭酸塩、ビス重炭酸塩、カルバミン酸塩、及びビスカルバミン酸塩からなる群より選択される１種以上の形態であり、
　前記濃縮工程において、前記塩の形態のジアミンを、遊離塩基及び二酸化炭素に転換させ、二酸化炭素を分離させる、［１２］～［２０］のいずれか１項に記載の製造方法；
［２２］前記有機溶媒が、ヘキサン、ブタノール、及び２－エチル－１－ヘキサノールからなる群より選択される１種以上である、［１８］～［２１］のいずれか１項に記載の製造方法；
［２３］前記ジアミンが、前記組換え微生物による、糖、二酸化炭素、合成ガス、メタノール、及びアミノ酸のうちの少なくとも１つ以上の発酵により生成されたものである、［１２］～［２２］のいずれか１項に記載の製造方法；
［２４］前記分離した前記無機塩を含む水相を培養液として再使用することを含む、［１７］～［２３］のいずれか１項に記載の製造方法；
［２５］得られたジアミンを精製する精製工程を含む、［１２］～［２４］のいずれか１項に記載の製造方法。 Thus, the present invention provides:
[1] A halophilic and/or alkalophilic recombinant microorganism having diamine-producing ability,
The diamine is represented by the formula: NH 2 CH 2 (CH 2 ) n CH 2 NH 2 (wherein n is an integer from 0 to 10),
A recombinant microorganism in which a halophilic and/or alkalophilic host microorganism has been modified to have diamine-producing ability;
[2] The recombinant microorganism according to [1], wherein n in the formula is 3 or 4;
[3] The recombinant microorganism of [2], wherein the host microorganism has been modified by one or more genetic manipulations to induce overproduction of L-lysine decarboxylase (EC 4.1.1.18);
[4] The genetic manipulation includes the following (A), (B), (C), (D) and (E):
(A) an operation of introducing an exogenous gene encoding the L-lysine decarboxylase into the host microorganism;
(B) increasing the copy number of the endogenous gene for L-lysine decarboxylase in the host microorganism;
(C) an operation of introducing a mutation into the expression control region of the endogenous gene of the L-lysine decarboxylase in the host microorganism;
(D) an operation of replacing the expression regulatory region of the endogenous gene of the L-lysine decarboxylase in the host microorganism with an exogenous regulatory region capable of high expression; and (E) the L-lysine decarboxylase in the host microorganism. The recombinant microorganism of [3], wherein one or more manipulations are selected from the group consisting of manipulations that delete the regulatory region of the endogenous gene for carboxylase;
[5] containing a nucleotide sequence having 85, 90, 92, 95, 98 or 99% or more homology with the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or the amino acid sequence shown in SEQ ID NO: 1 or 3 The recombinant microorganism according to any one of [2] to [4], comprising a nucleotide sequence having 85, 90, 92, 95, 98 or 99% or more homology with the nucleotide sequence encoding
[6] The recombinant microorganism according to any one of [2] to [5], which is further modified by mutation or gene recombination to improve lysine-producing ability;
[7] The mutagenesis or genetic recombination is performed by at least aspartokinase III (EC 2.7.2.4) and 4-hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7) The recombinant microorganism according to [6], which is an operation to release feedback inhibition on one side;
[8] The host microorganism is selected from the group consisting of Bacillus pseudofirmus, Bacillus halodurans, and Bacillus marmarensis, [1]-[7 ] The recombinant microorganism according to any one of
[9] The recombinant microorganism according to any one of [1] to [8], wherein the host microorganism is Bacillus pseudofirmus;
[10] Using the recombinant microorganism according to any one of [1] to [9], formula: NH 2 CH 2 (CH 2 ) n CH 2 NH 2 (wherein n is 0 to 10 is an integer);
[11] The production method according to [10], wherein n in the formula is 3 or 4;
[12] The production method according to [10] or [11], comprising a culturing step of obtaining a diamine-containing culture solution by culturing the recombinant microorganism in a medium containing 5% by mass or more of an inorganic salt;
[13] a culturing step of culturing the recombinant microorganism to obtain a culture solution containing bacterial cells;
a reaction step of obtaining a reaction solution containing 1,5-pentanediamine by contacting the culture solution and/or the bacterial cells with an aqueous solution containing 5% by mass or more of an inorganic salt and lysine, [10] ~ The production method according to any one of [12];
[14] The production method according to [12] or [13], comprising a removal step of removing the recombinant microorganism from the culture solution or reaction solution;
[15] The production method according to [14], including a concentration step of concentrating the culture solution or reaction solution;
[16] The production method according to [15], including a pH adjustment step of adjusting the pH of the concentrated culture medium or reaction liquid to 12 or higher after concentrating the culture medium or reaction liquid in the concentration step;
[17] The production according to any one of [12] to [16], including a separation step of phase-separating a phase containing a diamine and an aqueous phase containing the inorganic salt from the culture solution or reaction solution. Method;
[18] Any one of [12] to [17], wherein in the separation step, an organic solvent is added to the culture solution or the reaction solution to cause phase separation into a phase containing a diamine and an organic solvent and an aqueous phase. The manufacturing method according to paragraph;
[19] The production method according to [17] or [18], wherein no alkali compound is added in the separation step;
[20] The production method according to any one of [12] to [19], wherein the inorganic salt is sodium carbonate and/or sodium sulfate;
[21] One or more forms in which the diamine produced in the culture step and/or the reaction step is selected from the group consisting of carbonate, bicarbonate, bisbicarbonate, carbamate, and biscarbamate and
The production method according to any one of [12] to [20], wherein in the concentration step, the diamine in the form of a salt is converted into a free base and carbon dioxide, and the carbon dioxide is separated;
[22] The production method according to any one of [18] to [21], wherein the organic solvent is one or more selected from the group consisting of hexane, butanol, and 2-ethyl-1-hexanol. ;
[23] of [12] to [22], wherein the diamine is produced by fermentation of at least one or more of sugar, carbon dioxide, syngas, methanol, and amino acids by the recombinant microorganism. The manufacturing method according to any one of the items;
[24] The production method according to any one of [17] to [23], which comprises reusing the separated aqueous phase containing the inorganic salt as a culture solution;
[25] The production method according to any one of [12] to [24], including a purification step of purifying the obtained diamine.
　さらに本発明者らは、上記の更なる課題を解決するために、鋭意検討を行った。具体的には、本発明者らは、上記のように、ジアミン生産プロセスの簡略化及び排水処理コストの削減するために、好塩性および／または好アルカリ性の組換え微生物の使用を見出し、宿主微生物として好塩性および／または好アルカリ性の組換え微生物を使用することで、１，５－ペンタメチレンジアミンの生産時のｐＨ制御工程の簡略化、および、１，５－ペンタメチレンジアミンの遊離工程で発生する高濃度塩含有水の培地としての再利用を可能にしたが、好塩性および／または好アルカリ性の組換え微生物による１，５－ペンタメチレンジアミンの生産過程において、１，５－ペンタメチレンジアミンのＮ末端のアミノ基がアセチル化されたＮ－アセチルカダベリンが副生することが見出された。 Furthermore, the present inventors conducted intensive studies in order to solve the above further problems. Specifically, the present inventors discovered the use of halophilic and/or alkalophilic recombinant microorganisms to simplify the diamine production process and reduce wastewater treatment costs, as described above, and By using a halophilic and/or alkalophilic recombinant microorganism as a microorganism, simplification of the pH control step during the production of 1,5-pentamethylenediamine and the liberation step of 1,5-pentamethylenediamine. However, in the process of producing 1,5-pentamethylenediamine by halophilic and/or alkalophilic recombinant microorganisms, 1,5-pentamethylenediamine It was found that N-acetylcadaverine, in which the N-terminal amino group of methylenediamine was acetylated, was produced as a by-product.
　１，５－ペンタメチレンジアミンのＮ－アセチル化は、微生物細胞に内在するアセチルトランスフェラーゼによって触媒されている。コリネ菌においては、１，５－ペンタメチレンジアミンのＮ－アセチル化酵素が同定され、当該酵素をコードする遺伝子を破壊することによって、１，５－ペンタメチレンジアミンのＮ－アセチル化を抑制している（特許文献９および特許第５９６０６０４号公報）。 The N-acetylation of 1,5-pentamethylenediamine is catalyzed by an acetyltransferase endogenous to microbial cells. In Corynebacterium, an N-acetylation enzyme of 1,5-pentamethylenediamine has been identified, and the N-acetylation of 1,5-pentamethylenediamine can be suppressed by disrupting the gene encoding the enzyme. (Patent Document 9 and Japanese Patent No. 5960604).
　しかし、好塩性および／または好アルカリ性の組換え微生物のゲノム情報にて、相同性検索を実施した結果、当該遺伝子のホモログが存在しないことが判明した。そのため、好塩性および／または好アルカリ性の組換え微生物において、コリネ菌とは系統学的に異なる、いずれかのアセチルトランスフェラーゼが１，５－ペンタメチレンジアミンのアセチル化を触媒していると予測された。 However, as a result of performing a homology search on the genome information of halophilic and/or alkalophilic recombinant microorganisms, it was found that there was no homologue of the gene. Therefore, in halophilic and/or alkalophilic recombinant microorganisms, it is predicted that either acetyltransferase, which is phylogenetically different from Corynebacterium, catalyzes the acetylation of 1,5-pentamethylenediamine. rice field.
　そこで、好塩性および／または好アルカリ性の組換え微生物による１，５－ペンタメチレンジアミン生産において、ジアミンのアセチル化を抑制するためには、まずは、１，５－ペンタメチレンジアミンのアセチル化を触媒している酵素を特定する必要があった。 Therefore, in the production of 1,5-pentamethylenediamine by a halophilic and/or alkalophilic recombinant microorganism, in order to suppress the acetylation of diamine, first, the acetylation of 1,5-pentamethylenediamine is catalyzed. It was necessary to identify the enzymes that
　上記鋭意検討の結果、本発明者らは、好塩性および／または好アルカリ性の宿主微生物のゲノム配列から１，５－ペンタメチレンジアミンのアセチル化を触媒する酵素をコードする遺伝子を特定することに成功し、当該遺伝子に改変を行い、好塩性および／または好アルカリ性の宿主微生物において、副生物であるＮ－アセチルジアミン生産を抑制するに至った。 As a result of the above intensive studies, the present inventors determined to identify a gene encoding an enzyme that catalyzes the acetylation of 1,5-pentamethylenediamine from the genome sequences of halophilic and/or alkalophilic host microorganisms. Successfully, modifications have been made to the gene leading to suppression of by-product N-acetyldiamine production in halophilic and/or alkalophilic host microorganisms.
　すなわち本発明は、さらに以下を提供する：
［２６］ジアミン生産能を有する、好塩性および／または好アルカリ性の組換え微生物であって、
　ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成するＮ－アセチル化酵素を抑制する１以上の遺伝子改変を含む、組換え微生物；
［２７］前記遺伝子改変が、
・前記Ｎ－アセチル化酵素をコードする内因性遺伝子の発現を抑制する改変であるか、または、
・当該Ｎ－アセチル化酵素の活性が低下する改変である、［２６］に記載の組換え微生物；
［２８］Ｎ－アセチルジアミン化合物の生産能が、前記遺伝子改変を含まない非変異株の当該生産能と比較して、抑制されているか又は消失している、［２６］または［２７］に記載の組換え微生物；
［２９］前記Ｎ－アセチル化酵素をコードする遺伝子がｙｊｂＣ遺伝子である、［２６］～［２８］のいずれか１項に記載の組換え微生物；
［３０］前記Ｎ－アセチル化酵素が、
　（Ａ）（Ａ－１）配列番号２３に示されるアミノ酸配列からなるか、
　（Ａ－２）配列番号２３に示されるアミノ酸配列と８５％以上、９０％以上、９５％以上、９７％以上、９８％以上または９９％以上の配列同一性を有するアミノ酸配列からなり、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するか、もしくは
　（Ａ－３）配列番号２３に示されるアミノ酸配列に対して１～１０個、１～７個、１～５個、または１～３個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するか、または
　（Ｂ）（Ｂ－１）配列番号２４に示される塩基配列からなるＤＮＡ、
　（Ｂ－２）配列番号２４に示される塩基配列に相補的な塩基配列を有するＤＮＡと緊縮条件下でハイブリダイズし、かつ、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するタンパク質をコードするＤＮＡ、
　（Ｂ－３）配列番号２４に示される塩基配列と８５％以上、９０％以上、９５％以上、９７％以上、９８％以上または９９％以上の配列同一性を有する塩基配列からなり、かつ、
ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するタンパク質をコードするＤＮＡ、
　（Ｂ－４）配列番号２４に示される塩基配列によりコードされるタンパク質のアミノ酸配列に対して１～１０個、１～７個、１～５個、または１～３個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなるタンパク質をコードするＤＮＡであって、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するタンパク質をコードするＤＮＡ、もしくは
　（Ｂ－５）配列番号２４に示される塩基配列の縮重異性体からなるＤＮＡ
にコードされる、［２６］～［２９］のいずれか１項に記載の組換え微生物；
［３１］好塩性および／または好アルカリ性の宿主微生物に対して、前記Ｎ－アセチル化酵素を抑制する１以上の遺伝子改変が行われた、［２６］～［３０］のいずれか１項に記載の組換え微生物；
［３２］前記宿主微生物が、バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｅｕｄｏｆｉｒｍｕｓ）、バチルス・ハロデュランス(Ｂａｃｉｌｌｕｓ　ｈａｌｏｄｕｒａｎｓ)およびバチルス・マーマエンシス(Ｂａｃｉｌｌｕｓ　ｍａｒｍａｒｅｎｓｉｓ)からなる群より選択される、［２６］～［３１］のいずれか１項に記載の組換え微生物；
［３３］前記ジアミン化合物が、式：ＮＨ２ＣＨ２(ＣＨ２)ｎＣＨ２ＮＨ２（式中、ｎは、０、１、２、３、４、５、６、７、８、９または１０である。）で表される、［２６］～［３２］のいずれか１項に記載の組換え微生物；
［３４］前記ジアミン化合物がカダベリンである、［２６］～［３３］のいずれか１項に記載の組換え微生物；
［３５］［２６］～［３４］のいずれか１項記載の組換え微生物を培養して、当該組み換え微生物の培養物および／または培養物の抽出物を得る培養工程を含む、ジアミン化合物の製造方法；
［３６］前記培養物および／または前記培養物の抽出物を、基質化合物と混合して混合液を得る混合工程をさらに含む、［３５］に記載の製造方法；
［３７］前記培養物または前記混合液から、ジアミン化合物を回収する回収工程をさらに含む、［３５］または［３６］に記載の製造方法。 Thus, the present invention further provides:
[26] A halophilic and/or alkalophilic recombinant microorganism capable of producing diamine,
A recombinant microorganism comprising one or more genetic modifications that inhibit an N-acetyltransferase that N-acetylates a diamine compound to produce an N-acetyldiamine compound;
[27] The genetic modification is
- modification that suppresses the expression of the endogenous gene encoding the N-acetyltransferase, or
- The recombinant microorganism of [26], which is a modification that reduces the activity of the N-acetyltransferase;
[28] according to [26] or [27], wherein the ability to produce an N-acetyldiamine compound is suppressed or eliminated compared to the production ability of the non-mutant strain that does not contain the genetic modification; a recombinant microorganism of
[29] The recombinant microorganism according to any one of [26] to [28], wherein the gene encoding the N-acetyltransferase is the yjbC gene;
[30] The N-acetyltransferase is
(A) (A-1) consists of the amino acid sequence shown in SEQ ID NO: 23,
(A-2) A diamine compound consisting of an amino acid sequence having 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23. or (A-3) 1 to 10, 1 to 7, 1 to 5 relative to the amino acid sequence shown in SEQ ID NO: 23 consisting of an amino acid sequence in which 1 or 1 to 3 amino acids are deleted, substituted, inserted and/or added, and having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound, or (B) (B-1) DNA consisting of the base sequence shown in SEQ ID NO: 24;
(B-2) an enzyme that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 24 and N-acetylates a diamine compound to produce an N-acetyldiamine compound DNA encoding a protein having activity;
(B-3) consists of a base sequence having 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the base sequence shown in SEQ ID NO: 24, and
DNA encoding a protein having an enzymatic activity that N-acetylates a diamine compound to produce an N-acetyldiamine compound;
(B-4) deletion of 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids from the amino acid sequence of the protein encoded by the nucleotide sequence shown in SEQ ID NO: 24; A DNA encoding a protein consisting of a substituted, inserted and/or added amino acid sequence, the DNA encoding a protein having the enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound, or B-5) DNA consisting of a degenerate isomer of the base sequence shown in SEQ ID NO: 24
The recombinant microorganism according to any one of [26] to [29], encoded in;
[31] Any one of [26] to [30], wherein the halophilic and/or alkalophilic host microorganism is subjected to one or more genetic modifications that suppress the N-acetyltransferase a recombinant microorganism as described;
[32] The host microorganism is selected from the group consisting of Bacillus pseudofirmus, Bacillus halodurans and Bacillus marmarensis, [26]-[31] Recombinant microorganism according to any one of
[33] The diamine compound has the formula: NH2CH2 ( CH2 ) nCH2NH2 (wherein n is 0, 1, 2 , 3, 4 , 5, 6, 7, 8, 9 or 10.) Recombinant microorganism according to any one of [26] to [32];
[34] The recombinant microorganism according to any one of [26] to [33], wherein the diamine compound is cadaverine;
[35] Production of a diamine compound, comprising a culturing step of culturing the recombinant microorganism according to any one of [26] to [34] to obtain a culture of the recombinant microorganism and/or an extract of the culture. Method;
[36] The production method according to [35], further comprising a mixing step of mixing the culture and/or the culture extract with a substrate compound to obtain a mixed solution;
[37] The production method according to [35] or [36], further comprising a recovery step of recovering a diamine compound from the culture or the mixed solution.
　本発明により、ジアミンの生産において、プロセスの簡略化及び排水処理コストの削減の少なくとも一方を実現することができる。 According to the present invention, at least one of process simplification and wastewater treatment cost reduction can be realized in diamine production.
　また、本発明により、好塩性および／または好アルカリ性のジアミン生産菌を用いたジアミンの生産において、副生物であるＮ－アセチルジアミンの生産を抑制することができる。 In addition, according to the present invention, the production of N-acetyldiamine, which is a by-product, can be suppressed in diamine production using halophilic and/or alkalophilic diamine-producing bacteria.
図１は、ヘキサメチレンジアミン生産経路の一例を示す図である。FIG. 1 is a diagram showing an example of a hexamethylenediamine production pathway. 図２Ａは、各酵素のアミノ酸配列を示す図である。FIG. 2A shows the amino acid sequences of each enzyme. 図２Ｂは、各酵素のアミノ酸配列を示す図である。FIG. 2B is a diagram showing the amino acid sequence of each enzyme. 図３は、バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｕｅｄｏｆｉｒｍｕｓ）のｙｊｂＣ酵素のアミノ酸配列（配列番号２３）を示す図である。FIG. 3 shows the amino acid sequence (SEQ ID NO: 23) of the yjbC enzyme of Bacillus pseudofirmus. 図４は、バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｕｅｄｏｆｉｒｍｕｓ）ｙｊｂＣ遺伝子の塩基配列（配列番号２４）を示す図である。FIG. 4 shows the nucleotide sequence (SEQ ID NO: 24) of the Bacillus pseudofirmus yjbC gene. 図５Ａは、プライマーの塩基配列を示す図である。FIG. 5A is a diagram showing base sequences of primers. 図５Ｂは、プライマーの塩基配列を示す図である。FIG. 5B is a diagram showing base sequences of primers.
　以下、本発明を実施するための形態について詳細に説明する。なお、本発明は、以下の実施形態に限定されるものではなく、その要旨の範囲内で種々変形して実施することができる。また、本明細書に記述されているＤＮＡの取得、ベクターの調製および形質転換等の遺伝子操作は、特に明記しない限り、Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ　４ｔｈ　Ｅｄｉｔｉｏｎ（Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ，　２０１２）、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ（Ｇｒｅｅｎｅ　Ｐｕｂｌｉｓｈｉｎｇ　Ａｓｓｏｃｉａｔｅｓ　ａｎｄ　Ｗｉｌｅｙ－Ｉｎｔｅｒｓｃｉｅｎｃｅ）、および、遺伝子工学実験ノート（羊土社　田村隆明）等の公知の文献に記載されている方法により行うことができる。本明細書において特に断りのない限りヌクレオチド配列は５’方向から３’方向に向けて記載される。本明細書において、「ポリペプチド」および「タンパク質」の語は、互換可能に使用される。また、「遺伝子組換え微生物」は、単に「組換え微生物」とも称される。 Hereinafter, the embodiments for carrying out the present invention will be described in detail. It should be noted that the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the gist of the present invention. In addition, unless otherwise specified, genetic manipulations such as DNA acquisition, vector preparation and transformation described herein are described in Molecular Cloning 4th Edition (Cold Spring Harbor Laboratory Press, 2012), Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience) and Genetic Engineering Experimental Notes (Takaaki Tamura, Yodosha). Nucleotide sequences are written in the 5' to 3' direction unless otherwise indicated herein. As used herein, the terms "polypeptide" and "protein" are used interchangeably. A "genetically modified microorganism" is also simply referred to as a "recombinant microorganism".
　本明細書において、「～」を用いて示された数値範囲は、「～」の前後に記載される数値をそれぞれ最小値及び最大値として含む範囲を示す。本明細書に段階的に記載されている数値範囲において、ある段階の数値範囲の上限値又は下限値は、他の段階の数値範囲の上限値又は下限値と任意に組み合わせることができる。 In this specification, a numerical range indicated using "-" indicates a range that includes the numerical values before and after "-" as the minimum and maximum values, respectively. In the numerical ranges described stepwise in this specification, the upper limit value or lower limit value of the numerical range in one step can be arbitrarily combined with the upper limit value or lower limit of the numerical range in another step.
　本明細書において、「内因」または「内因性」の用語は、遺伝子組換えによる改変がなされていない宿主微生物が、言及している遺伝子ないしはそれによりコードされるタンパク質（典型的には酵素）を、当該宿主細胞内で優位な生化学的反応を進行させ得る程度に機能的に発現しているかどうかに関わらず、宿主微生物が有していることを意味するために用いられる。「内在性」および「内因性」の用語は、本明細書において互換可能に用いられる。 As used herein, the term "endogenous" or "endogenous" means that a host microorganism that has not been modified by genetic recombination has the gene referred to or the protein (typically an enzyme) encoded by it. , is used to mean that the host microorganism has whether or not it is functionally expressed to the extent that it can drive dominant biochemical reactions within the host cell. The terms "endogenous" and "endogenous" are used interchangeably herein.
　本明細書において、「外来」または「外来性」の用語は、遺伝子組換え前の宿主微生物が導入されるべき遺伝子を有していない場合、その遺伝子による酵素を実質的に発現していない場合、及び異なる遺伝子により当該酵素のアミノ酸配列をコードしているが、遺伝子組換え後に匹敵する内因性酵素活性を発現しない場合において、遺伝子または核酸配列を宿主に導入することを意味するために用いられる。「外来性」および「外因性」の用語は、本明細書において互換可能に用いられる。 As used herein, the term "foreign" or "exogenous" is used when the host microorganism before genetic recombination does not have the gene to be introduced, when the enzyme by the gene is not substantially expressed , and to denote the introduction of a gene or nucleic acid sequence into a host where a different gene encodes the amino acid sequence of the enzyme but does not express comparable endogenous enzymatic activity after genetic recombination . The terms "exogenous" and "exogenous" are used interchangeably herein.
　本明細書において、微生物に関し、「ジアミン生産能を有する」とは、その微生物を使用したジアミン生産のプロセスのいずれかの段階において、ジアミンが生産される微生物をいう。具体的には、当該微生物の培養によって得られた培養液にジアミンが含まれていてもよいし、培地にジアミンの前駆体、例えば、カルボン酸化合物、アルデヒド化合物および／またはカルボニル化合物を添加し、ジアミン前駆体をジアミンに変換する微生物を培養することによってジアミンを生産してもよい。「ジアミン生産能を有する微生物」には、これらの性質の１つまたは複数を有する微生物が含まれる。 As used herein, with respect to microorganisms, "having diamine-producing ability" refers to microorganisms that produce diamine in any stage of the diamine-producing process using the microorganism. Specifically, the culture solution obtained by culturing the microorganism may contain a diamine, or a diamine precursor such as a carboxylic acid compound, an aldehyde compound and/or a carbonyl compound is added to the medium, Diamines may be produced by culturing microorganisms that convert diamine precursors to diamines. A "microorganism capable of producing diamines" includes microorganisms having one or more of these properties.
　さらに、組換え微生物に関し、「ジアミン生産能を有する」とは、当該微生物がジアミンの生産経路を有することを意味する。本明細書において、ある化合物に関し、微生物が「生産経路を有する」とは、当該微生物が、その化合物の生産経路の各反応段階が進行するのに十分な量の酵素を発現し、その化合物を生合成可能であることを意味する。本発明の組換え微生物は、本来ジアミンを生産する能力を有する宿主微生物を用いたものであってもよく、本来はジアミンを生産する能力を有さない、好塩性および／または好アルカリ性の宿主微生物に対して、ジアミン生産能を有するように改変を行ったものであってもよい。 Furthermore, with regard to recombinant microorganisms, "having diamine-producing ability" means that the microorganism has a diamine production pathway. As used herein, with respect to a certain compound, a microorganism "has a production pathway" means that the microorganism expresses a sufficient amount of an enzyme for each reaction step in the production pathway of the compound to proceed, and produces the compound. It means that it can be biosynthesized. The recombinant microorganism of the present invention may be one using a host microorganism that originally has the ability to produce diamine, or a halophilic and/or alkalophilic host that does not originally have the ability to produce diamine. Microorganisms that have been modified to have diamine-producing ability may also be used.
　本発明に関連して、ジアミン化合物は、式：ＮＨ２ＣＨ２(ＣＨ２)ｎＣＨ２ＮＨ２で表される。式中、ｎは、例えば０、１、２、３、４、５、６、７、８、９または１０、好ましくは２、３、４または５、より好ましくは２、３または４、特に好ましくは３または４である。 In the context of the present invention, diamine compounds are represented by the formula : NH2CH2 ( CH2 ) nCH2NH2 . wherein n is, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 2, 3, 4 or 5, more preferably 2, 3 or 4, particularly preferably is 3 or 4.
　ジアミン化合物の例としては、エチレンジアミン、プロピレンジアミン、テトラメチレンジアミン（例えばプトレッシン）、ペンタメチレンジアミン（例えばカダベリン）、ヘキサメチレンジアミン、およびへプタメチレンジアミンが挙げられる。好ましい一態様において、ジアミン化合物は、ヘキサメチレンジアミンまたはカダベリンである。 Examples of diamine compounds include ethylenediamine, propylenediamine, tetramethylenediamine (eg putrescine), pentamethylenediamine (eg cadaverine), hexamethylenediamine, and heptamethylenediamine. In one preferred embodiment, the diamine compound is hexamethylenediamine or cadaverine.
＜１＞発明Ａ：ジアミン生産能を有する組換え微生物およびジアミンの製造方法
　本発明にかかる組換え微生物は、ジアミン生産能を有する微生物であり、具体的には、好塩性及び好アルカリ性の少なくとも一方を有する宿主微生物に対して、ジアミン生産能を有するように改変を行ったものである。 <1> Invention A: Recombinant microorganism having diamine-producing ability and method for producing diamine A host microorganism having one side is modified so as to have diamine-producing ability.
　自然界には、ｐＨ９以上のアルカリ環境でも生育することができる好アルカリ性の性質、０．２Ｍ以上のＮａＣｌ濃度の環境でも生育することができる好塩性の性質、またはこれら両者の性質を併せ持つ微生物が存在する。本発明者らは、上記の好アルカリ性微生物をジアミンの生産に使用することができれば、酵素変換工程あるいは培養工程にて生成物濃度向上によりｐＨが上昇しても生育可能であるため、酸溶液添加によるｐＨ調整を省略することができると考えた。また、上記の好塩性微生物をジアミンの生産に使用することができれば、遊離工程にて発生する高濃度塩含有水を培地として再利用することができると考えた。なお、好塩性及び好アルカリ性の少なくとも一方の性質を持つ微生物を、ジアミン生産に適用した例はこれまでに報告されてない。 In the natural world, there are microorganisms that have alkalophilic properties that allow them to grow in an alkaline environment with a pH of 9 or higher, halophilic properties that allow them to grow in an environment with a NaCl concentration of 0.2M or higher, or those that have both of these properties. exist. The present inventors believe that if the above-mentioned alkalophilic microorganisms can be used to produce diamines, they can grow even if the pH rises due to an increase in product concentration in the enzymatic conversion process or culture process. It was thought that the pH adjustment by could be omitted. Also, if the halophilic microorganisms described above can be used for the production of diamine, the water containing high concentrations of salts generated in the separation process can be reused as a medium. There have been no reports of applications of microorganisms having at least one of halophilicity and alkalophilicity to diamine production.
　本発明者らは、好塩性及び好アルカリ性の少なくとも一方の性質を持つ微生物を宿主微生物として使用し、ジアミン生産能を有するように改変を試みたところ、得られた組換え微生物によれば、プロセスの簡略化及び排水処理コストの削減の少なくとも一方が可能となることを見出した。 The present inventors used a microorganism having at least one of halophilic and alkalophilic properties as a host microorganism and attempted to modify it so as to have diamine-producing ability. According to the obtained recombinant microorganism, It has been found that at least one of process simplification and wastewater treatment cost reduction is possible.
　すなわち、本発明者らは、好塩性および／または好アルカリ性微生物にジアミン生産能を付与することに成功し、かかる微生物を用いることで、酵素変換工程または培養工程等において生成物濃度上昇によりｐＨが上昇した場合でも、酸溶液添加等のｐＨ調整を行うことなく微生物を生育させることができることと、更に好適な態様においては、ジアミンを単離する際に発生する高濃度塩含有水を培地として再利用することができることを見出した。 That is, the present inventors have succeeded in imparting diamine-producing ability to halophilic and/or alkalophilic microorganisms, and by using such microorganisms, pH Even when the pH is increased, the microorganism can be grown without adjusting the pH such as adding an acid solution, and in a more preferred embodiment, the high-concentration salt-containing water generated when isolating the diamine is used as a medium. We have found that it can be reused.
　本発明の実施形態の組換え微生物の作製において、例えば、好塩性及び好アルカリ性の少なくとも一方の性質を持つ宿主微生物の細胞内に外来の遺伝子を導入する技術、ゲノム配列に任意の外来の遺伝子配列を導入する技術、またはゲノム配列から不要な遺伝子配列を取り除く技術を利用することができる。 In the preparation of the recombinant microorganisms of the embodiments of the present invention, for example, techniques for introducing foreign genes into the cells of host microorganisms having at least one of halophilic and alkalophilic properties, any foreign gene in the genome sequence Techniques for introducing sequences or removing unwanted gene sequences from genomic sequences can be used.
　すなわち、以下のような技術が挙げられる。
（１）好塩性、好アルカリ性、または両方の性質を持つ宿主微生物を、当該宿主細胞内で発現可能なプロモーターとジアミン生産に関わる遺伝子との組み合わせにより形質転換する技術、
（２）前技術が、好塩性、好アルカリ性、または両方の性質を持つ微生物の細胞内で安定的に複製されるプラスミドベクターを使用することを特徴とする、上記（１）の技術、
（２’）前技術の対象となるプラスミドベクターがｐＵＢ１１０である、上記（２）の技術、
（３）前技術が、好塩性、好アルカリ性、または両方の性質を持つ微生物の細胞内で所望する形質が安定的に発現することを特徴とする、上記（１）の技術、
（４）温度感受性プラスミドベクターとネガティブセレクションを組み合わせた染色体上の遺伝子配列を任意に改変する技術、
（５）前技術が、３７℃以上の温度において微生物が保有するプラスミドの複製を停止させる、もしくは保有しているプラスミドのコピー数が減少させることを特徴とする、上記（４）の技術、
（６）前技術が、スクロースおよび４－クロロフェニルアラニンといった物質が存在する条件において特定の遺伝子を保有する微生物の増殖が阻害されることで、特定の遺伝子を保有しない微生物をセレクション（ネガティブセレクション）することを特徴とする、上記の（４）の技術、
（６’）前技術の対象となる特定の遺伝子が、レバンスクラーゼ遺伝子（ｓａｃＢ遺伝子）である、上記（６）の技術、
（６”）前技術の対象となる特定の遺伝子が、塩基配列に変異を導入した宿主由来のフェニルアラニンｔＲＮＡ合成酵素αサブユニット遺伝子（ｐｈｅＳ遺伝子）である、上記（６）の技術。 That is, the following techniques are mentioned.
(1) A technique of transforming a host microorganism having halophilicity, alkalophilicity, or both properties by combining a promoter capable of being expressed in the host cell and a gene involved in diamine production,
(2) The technology of (1) above, wherein the prior technology uses a plasmid vector that is stably replicated in cells of microorganisms having halophilicity, alkalophilicity, or both properties;
(2') The technology of (2) above, wherein the plasmid vector to be the subject of the prior technology is pUB110,
(3) The technology of (1) above, wherein the prior art is characterized by stably expressing desired traits in cells of microorganisms having halophilicity, alkalophilicity, or both properties;
(4) Techniques for arbitrarily modifying gene sequences on chromosomes by combining temperature-sensitive plasmid vectors and negative selection,
(5) The technology of (4) above, wherein the prior art stops replication of the plasmid carried by the microorganism or reduces the copy number of the plasmid carried by the microorganism at a temperature of 37° C. or higher;
(6) The prior art selects (negative selection) microorganisms that do not possess a specific gene by inhibiting the growth of microorganisms that possess a specific gene in the presence of substances such as sucrose and 4-chlorophenylalanine. The technology of (4) above, characterized by
(6′) The technology of (6) above, wherein the specific gene targeted by the prior technology is the levansucrase gene (sacB gene);
(6″) The technique of (6) above, wherein the specific gene targeted by the prior art is a host-derived phenylalanine tRNA synthetase α-subunit gene (pheS gene) into which a mutation has been introduced in the base sequence.
　本発明の好適な態様の一つにおいては、高発現プロモーターと外来遺伝子をプラスミドベクターに保有させ、好塩性、好アルカリ性、または両方の性質を持つ微生物の細胞内に導入することにより、外来遺伝子が安定的に高発現する。また、別の好適な態様では、温度感受性のプラスミドベクターとネガティブセレクションを組み合わせた技術により、好塩性、好アルカリ性、または両方の性質を持つ微生物が保持するゲノム配列を任意に編集する。当該技術を利用して遺伝子導入またはゲノム配列が編集される細胞株は、通常の意味での野生型の株であってよく、或いは、その野生型の株に由来する栄養要求性変異株、抗生物質耐性変異株であってもよい。更に、本発明の宿主細胞として利用できる細胞株は、上記のような変異に関する各種マーカー遺伝子を有するように既に形質転換されていてもよい。これらの技術により、本発明の組換微生物の作製、維持および／または管理に有益な性質を提供できる。 In one of the preferred embodiments of the present invention, a plasmid vector carries a high-expression promoter and a foreign gene, and is introduced into the cells of microorganisms having halophilicity, alkalophilicity, or both, thereby exposing the foreign gene to is stably highly expressed. In another preferred embodiment, genome sequences possessed by halophilic, alkalophilic, or both halophilic and/or alkalophilic microorganisms are optionally edited using a combination of temperature-sensitive plasmid vectors and negative selection. Cell lines whose gene transfer or genome sequence is edited using this technology may be wild-type strains in the usual sense, or auxotrophic mutant strains derived from the wild-type strains, antibiotics It may be a substance-resistant mutant strain. Furthermore, cell lines that can be used as host cells of the present invention may already be transformed to have various marker genes for mutations as described above. These techniques can provide beneficial properties for the production, maintenance and/or management of the recombinant microorganisms of the invention.
　本発明に関連して、宿主微生物として利用できる好アルカリ性微生物とは、多様な分布を示す極限環境微生物の一種であり、ｐＨが９以上の環境下でも生育可能な微生物の総称である。これらは、ｐＨ９以上の環境でのみ生育できる絶対好アルカリ性微生物と、ｐＨ９以上に至適生育ｐＨを示すものの、ｐＨ９未満でも生育することができる通性好アルカリ性微生物に分類される。その中のいくつかはｐＨ１２以上の強アルカリ性環境でも生育することができる。これらのいずれもが、本発明の好アルカリ性微生物である。 In relation to the present invention, alkalophilic microorganisms that can be used as host microorganisms are a type of extremophilic microorganisms that exhibit diverse distributions, and are a general term for microorganisms that can grow even in an environment with a pH of 9 or higher. These are classified into obligate alkalophilic microorganisms that can grow only in an environment of pH 9 or higher, and facultative alkalophilic microorganisms that can grow at a pH of less than 9, although the optimum growth pH is at pH 9 or higher. Some of them can grow even in a strong alkaline environment with a pH of 12 or higher. All of these are alkalophilic microorganisms of the present invention.
　本発明に関連して、宿主微生物として利用できる好塩性微生物とは、高濃度の塩ストレスに対応できる微生物の総称である。これらは、最適増殖塩濃度による菌の分類では、塩化ナトリウム０～０．２Ｍが最適増殖塩濃度である非好塩菌、０．２～０．５Ｍが最適増殖塩濃度である低度好塩菌、０．５～２．５Ｍが最適増殖塩濃度である中度好塩菌、２．５～５．２Ｍが最適増殖塩濃度である高度好塩菌に分類される。これらのうち非好塩菌を除くいずれもが、本発明の好塩性微生物である。 In relation to the present invention, halophilic microorganisms that can be used as host microorganisms is a general term for microorganisms that can cope with high-concentration salt stress. These are non-halophilic bacteria whose optimum growth salt concentration is 0-0.2M sodium chloride, and low halophilic bacteria whose optimum growth salt concentration is 0.2-0.5M. Moderate halophiles whose optimum growth salt concentration is 0.5 to 2.5M, and high halophiles whose optimum growth salt concentration is 2.5 to 5.2M. All of these except non-halophilic bacteria are halophilic microorganisms of the present invention.
　本発明の宿主微生物として利用できる、好塩性、好アルカリ性、または両方の性質を持つ微生物には、様々な微生物が含まれるが、その非限定的な例としては、バチルス属、ハロモナス属、ハロバクテロイデス属、サリニバクター属、アルカリファイラス属、クロストリジウム属、アンエアロブランカ属の細菌等が挙げられる。本発明の宿主微生物は、好ましくは、バチルス属の細菌である。バチルス属の微生物の中でも、バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｅｕｄｏｆｉｒｍｕｓ）、バチルス・ハロデュランス(Ｂａｃｉｌｌｕｓ　ｈａｌｏｄｕｒａｎｓ)、バチルス・マーマエンシス(Ｂａｃｉｌｌｕｓ　ｍａｒｍａｒｅｎｓｉｓ)が好ましく、バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｕｅｄｏｆｉｒｍｕｓ）が更に好ましい。 Halophilic, alkalophilic, or both microorganisms that can be used as host microorganisms of the present invention include various microorganisms, non-limiting examples of which include Bacillus, Halomonas, Halo Bacteroides, Salinibacter, Alkalinephilus, Clostridium, Anaeroblanca, and the like. The host microorganism of the invention is preferably a bacterium of the genus Bacillus. Among microorganisms belonging to the genus Bacillus, Bacillus pseudofirmus, Bacillus halodurans, and Bacillus marmarensis are preferred, and Bacillus pseudofirmus is more preferred.
　本発明の好ましい態様では、宿主微生物において、新たなジアミン生合成経路を構築するための改変が行われている。例えば、ジアミンが１，５－ペンタメチレンジアミンである場合、組換え微生物は、新たな１，５－ペンタメチレンジアミン生合成経路を構築するために、１つ以上のＬ－リジンデカルボキシラーゼ（ＥＣ　４．１．１．１８）酵素遺伝子を上記宿主微生物の細胞に導入することにより得られる。ここで、Ｌ－リジンデカルボキシラーゼ（ＥＣ　４．１．１．１８）は、Ｌ－リジンを脱炭酸し、１，５－ペンタンジアミンを生成する反応を触媒する酵素である。或いは、組換え微生物は、上記酵素遺伝子配列を上記宿主微生物のゲノム配列に挿入することにより得られる。 In a preferred embodiment of the present invention, the host microorganism is modified to construct a new diamine biosynthetic pathway. For example, when the diamine is 1,5-pentamethylenediamine, the recombinant microorganism uses one or more L-lysine decarboxylase (EC 4 .1.1.18) obtained by introducing the enzyme gene into the cells of the host microorganism. Here, L-lysine decarboxylase (EC 4.1.1.18) is an enzyme that catalyzes the reaction of decarboxylating L-lysine to produce 1,5-pentanediamine. Alternatively, a recombinant microorganism is obtained by inserting the enzyme gene sequence into the genome sequence of the host microorganism.
　かかる操作により、得られた組換え微生物においてＬ－リジンデカルボキシラーゼ（ＥＣ　４．１．１．１８）の過剰生産が誘導される。すなわち、本発明にかかる組換え微生物は、Ｌ－リジンデカルボキシラーゼ（ＥＣ　４．１．１．１８）の過剰生産を誘導するように、１つ以上の遺伝子操作により改変されている。 Such manipulation induces overproduction of L-lysine decarboxylase (EC 4.1.1.18) in the resulting recombinant microorganism. That is, the recombinant microorganism according to the present invention is modified by one or more genetic manipulations so as to induce overproduction of L-lysine decarboxylase (EC 4.1.1.18).
　ジアミンが１，５－ペンタメチレンジアミンである場合、前記遺伝子操作が、例えば、下記（Ａ）、（Ｂ）、（Ｃ）、（Ｄ）及び（Ｅ）から成る群より選択される１以上の遺伝子操作であってよい。
（Ａ）宿主微生物に、Ｌ－リジンデカルボキシラーゼをコードする外来性遺伝子を導入する操作、
（Ｂ）宿主微生物内のＬ－リジンデカルボキシラーゼの内因性遺伝子のコピー数を増加させる操作、
（Ｃ）宿主微生物内のＬ－リジンデカルボキシラーゼの内因性遺伝子の発現調整領域に変異を導入する操作、
（Ｄ）宿主微生物内のＬ－リジンデカルボキシラーゼの内因性遺伝子の発現調整領域を、高発現可能な外来調整領域で置換する操作、および
（Ｅ）宿主微生物内のＬ－リジンデカルボキシラーゼの内因性遺伝子の調整領域を欠失させる操作。 When the diamine is 1,5-pentamethylenediamine, the genetic manipulation is, for example, one or more selected from the group consisting of (A), (B), (C), (D) and (E) below. It may be genetically engineered.
(A) an operation of introducing an exogenous gene encoding L-lysine decarboxylase into a host microorganism;
(B) an operation that increases the copy number of the endogenous gene for L-lysine decarboxylase in the host microorganism;
(C) an operation of introducing a mutation into the expression control region of the endogenous gene for L-lysine decarboxylase in the host microorganism;
(D) replacement of the expression regulatory region of the endogenous gene of L-lysine decarboxylase in the host microorganism with an exogenous regulatory region capable of high expression; and (E) endogenous L-lysine decarboxylase in the host microorganism. An operation that deletes the regulatory region of a gene.
　Ｌ－リジンデカルボキシラーゼ（ＥＣ　４．１．１．１８）の遺伝子の代表的なものとして、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）のｃａｄＡおよびｌｄｃＣが挙げられる。大腸菌ｃａｄＡ酵素のアミノ酸配列を配列番号１に、大腸菌ｃａｄＡの塩基配列を配列番号２に示す。また、大腸菌ｌｄｃＣ酵素のアミノ酸配列を配列番号３に、大腸菌ｌｄｃＣの塩基配列を配列番号４に示す。 Typical examples of genes for L-lysine decarboxylase (EC 4.1.1.18) include cadA and ldcC of Escherichia coli. The amino acid sequence of E. coli cadA enzyme is shown in SEQ ID NO:1, and the base sequence of E. coli cadA is shown in SEQ ID NO:2. In addition, the amino acid sequence of E. coli ldcC enzyme is shown in SEQ ID NO:3, and the base sequence of E. coli ldcC is shown in SEQ ID NO:4.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-I000003
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-I000003
　本発明の好ましい態様では、宿主微生物において、新たなジアミン生合成経路を構築するための改変が行われている。例えば、ジアミンがヘキサメチレンジアミンである場合、組換え微生物は、新たなヘキサメチレンジアミン生合成経路を構築するために、１つ以上の酵素遺伝子を上記宿主微生物の細胞に導入することにより得られる。或いは、組換え微生物は、上記酵素遺伝子配列を上記宿主微生物のゲノム配列に挿入することにより得られる。微生物が有しうるヘキサメチレンジアミン生産経路の一例を図１に示す。 In a preferred embodiment of the present invention, the host microorganism is modified to construct a new diamine biosynthetic pathway. For example, when the diamine is hexamethylenediamine, a recombinant microorganism is obtained by introducing one or more enzyme genes into the cells of the host microorganism to construct a new hexamethylenediamine biosynthetic pathway. Alternatively, a recombinant microorganism is obtained by inserting the enzyme gene sequence into the genome sequence of the host microorganism. FIG. 1 shows an example of a hexamethylenediamine production pathway that microorganisms can have.
　以下、各反応段階を触媒する酵素について例示する。 Below are examples of the enzymes that catalyze each reaction step.
　図１のステップＡの変換（スクシニルＣｏＡ：アセチルＣｏＡアシルトランスフェラーゼ、または３－オキソアジピルＣｏＡチオラーゼ）では、スクシニル－ＣｏＡとアセチル－ＣｏＡが縮合し、３－オキソアジピル－ＣｏＡへと変換される。本変換を触媒し得る酵素の例としては、β－ケトチオラーゼが挙げられる。例えば、ＥＣ　２．３．１．９（アセトアセチルＣｏＡチオラーゼ）およびＥＣ　２．３．１．１６（３－ケトアシル－ＣｏＡチオラーゼ）、ＥＣ　２．３．１．１７４（３－オキソアジピル－ＣｏＡチオラーゼ）といった群に分類される酵素は、本変換に対して活性を有し得る酵素として例示することができる。本発明で使用される酵素は、本変換に対する活性を有するものであれば限定されないが、例えば、配列番号１２に記載されるアミノ酸配列からなる大腸菌由来のＰａａＪが使用される（図２）。 In the conversion (succinyl-CoA: acetyl-CoA acyltransferase or 3-oxoadipyl-CoA thiolase) in step A of Fig. 1, succinyl-CoA and acetyl-CoA are condensed and converted to 3-oxoadipyl-CoA. Examples of enzymes that can catalyze this conversion include β-ketothiolase. For example, EC 2.3.1.9 (acetoacetyl-CoA thiolase) and EC 2.3.1.16 (3-ketoacyl-CoA thiolase), EC 2.3.1.174 (3-oxoadipyl-CoA thiolase) can be exemplified as enzymes that may have activity for this conversion. The enzyme used in the present invention is not limited as long as it has activity for this conversion. For example, E. coli-derived PaaJ consisting of the amino acid sequence set forth in SEQ ID NO: 12 is used (Fig. 2).
　図１のステップＢの変換（３－ヒドロキシアジピルＣｏＡデヒドロゲナーゼ）では、３－オキソアジピル－ＣｏＡが、３－ヒドロキシアジピル－ＣｏＡへと変換される。本変換を触媒し得る酵素の例としては、ＥＣ　１．１．１の群に分類されるオキシドレダクターゼが挙げられる。例えば、ＥＣ　１．１．１．３５（３－ヒドロキシアシル－ＣｏＡデヒドロゲナーゼ）、ＥＣ　１．１．１．３６（アセトアセチル－ＣｏＡデヒドロゲナーゼ）、ＥＣ　１．１．１．１５７（３－ヒドロキシブタノイル－ＣｏＡデヒドロゲナーゼ）、ＥＣ　１．１．１．２１１（長鎖３－ヒドロキシアシル－ＣｏＡデヒドロゲナーゼ）およびＥＣ　１．１．１．２５９（３－ヒドロキシピメロイル－ＣｏＡデヒドロゲナーゼ）といった群に分類される酵素は、本変換に対して活性を有し得る酵素として例示することができる。本発明で使用される酵素は、本変換に対する活性を有するものであれば限定されないが、例えば、配列番号１３に記載されるアミノ酸配列からなる大腸菌由来のＰａａＨが使用される（図２）。 In the conversion (3-hydroxyadipyl-CoA dehydrogenase) in step B of FIG. 1, 3-oxoadipyl-CoA is converted to 3-hydroxyadipyl-CoA. Examples of enzymes that can catalyze this conversion include oxidoreductases classified in group EC 1.1.1. For example, EC 1.1.1.35 (3-hydroxyacyl-CoA dehydrogenase), EC 1.1.1.36 (acetoacetyl-CoA dehydrogenase), EC 1.1.1.157 (3-hydroxybutanoyl -CoA dehydrogenase), EC 1.1.1.211 (long chain 3-hydroxyacyl-CoA dehydrogenase) and EC 1.1.1.259 (3-hydroxypymeloyl-CoA dehydrogenase). Enzymes can be exemplified as enzymes that may have activity for this conversion. The enzyme used in the present invention is not limited as long as it has activity for this conversion. For example, Escherichia coli-derived PaaH consisting of the amino acid sequence set forth in SEQ ID NO: 13 is used (Fig. 2).
　図１のステップＣの変換（３－ヒドロキシアジピルＣｏＡデヒドラターゼ）では、３－ヒドロキシアジピル－ＣｏＡが、２，３－デヒドロアジピル－ＣｏＡへと変換される。本変換を触媒し得る酵素の例としては、ＥＣ　４．２．１の群に分類されるヒドロリアーゼが挙げられる。例えば、ＥＣ　４．２．１．１７（エノイル－ＣｏＡヒドラターゼ）、ＥＣ　４．２．１．５５（３－ヒドロキシブタノイル－ＣｏＡデヒドラターゼ）およびＥＣ　４．２．１．７４（長鎖エノイル－ＣｏＡヒドラターゼ）といった群に分類される酵素は、本変換に対して活性を有し得る酵素として例示することができる。本発明で使用される酵素は、本変換に対する活性を有するものであれば限定されないが、例えば配列番号１４に記載されるアミノ酸配列からなる大腸菌由来のＰａａＦが使用される（図２）。 In the conversion (3-hydroxyadipyl-CoA dehydratase) in step C of FIG. 1, 3-hydroxyadipyl-CoA is converted to 2,3-dehydroadipyl-CoA. Examples of enzymes capable of catalyzing this conversion include hydrolyases classified in group EC 4.2.1. For example, EC 4.2.1.17 (enoyl-CoA hydratase), EC 4.2.1.55 (3-hydroxybutanoyl-CoA dehydratase) and EC 4.2.1.74 (long chain enoyl-CoA Enzymes classified into groups such as hydratases) can be exemplified as enzymes that may have activity for this conversion. The enzyme used in the present invention is not limited as long as it has activity for this conversion. For example, E. coli-derived PaaF consisting of the amino acid sequence shown in SEQ ID NO: 14 is used (Fig. 2).
　図１のステップＤの変換（２，３－デヒドロアジピルＣｏＡレダクターゼ）では、２，３－デヒドロアジピル－ＣｏＡが、アジピル－ＣｏＡへと変換される。本変換を触媒し得る酵素の例としては、ＥＣ　１．３．１の群に分類されるオキシドレダクターゼが挙げられる。例えば、ＥＣ　１．３．１．８（アシル－ＣｏＡデヒドロゲナーゼ（ＮＡＤＰ＋））、ＥＣ　１．３．１．９（エノイル－ＡＣＰレダクターゼ（ＮＡＤＨ））、ＥＣ　１．３．１．３８（トランス－２－エノイル－ＣｏＡレダクターゼ（ＮＡＤＰ＋））、ＥＣ　１．３．１．４４（トランス－２－エノイル－ＣｏＡレダクターゼ（ＮＡＤ＋））、ＥＣ　１．３．１．８６（クロトニル－ＣｏＡレダクターゼ）、ＥＣ　１．３．１．９３（長鎖アシル－ＣｏＡレダクターゼ）およびＥＣ　１．３．１．１０４（エノイル－ＡＣＰレダクターゼ（ＮＡＤＰＨ））といった群に分類される酵素は、本変換に対して活性を有し得る酵素として例示することができる。 In step D conversion (2,3-dehydroadipyl-CoA reductase) of FIG. 1, 2,3-dehydroadipyl-CoA is converted to adipyl-CoA. Examples of enzymes that can catalyze this conversion include oxidoreductases that fall into the group of EC 1.3.1. For example, EC 1.3.1.8 (acyl-CoA dehydrogenase (NADP + )), EC 1.3.1.9 (enoyl-ACP reductase (NADH)), EC 1.3.1.38 (trans- 2-enoyl-CoA reductase (NADP + )), EC 1.3.1.44 (trans-2-enoyl-CoA reductase (NAD + )), EC 1.3.1.86 (crotonyl-CoA reductase), Enzymes falling into the groups EC 1.3.1.93 (long-chain acyl-CoA reductase) and EC 1.3.1.104 (enoyl-ACP reductase (NADPH)) have activity towards this transformation. It can be exemplified as possible enzymes.
　本発明で使用される２，３－デヒドロアジピルＣｏＡレダクターゼは、例えば、Ｃａｎｄｉｄａ　ａｕｒｉｓ、Ｋｌｕｙｖｅｒｏｍｙｃｅｓ　ｍａｒｘｉａｎｕｓ、Ｐｉｃｈｉａ　ｋｕｄｒｉａｖｚｅｖｉｉ、Ｔｈｅｒｍｏｔｈｅｌｏｍｙｃｅｓ　ｔｈｅｒｍｏｐｈｉｌｕｓ、Ｔｈｅｒｍｏｔｈｉｅｌａｖｉｏｉｄｅｓ　ｔｅｒｒｅｓｔｒｉｓ、Ｃｈａｅｔｏｍｉｕｍ　ｔｈｅｒｍｏｐｈｉｌｕｍ、Ｐｏｄｏｓｐｏｒａ　ａｎｓｅｒｉｎａ、Ｐｕｒｐｕｒｅｏｃｉｌｌｉｕｍ　ｌｉｌａｃｉｎｕｍ、Ｐｙｒｅｎｏｐｈｏｒａ　ｔｅｒｅｓから選択されるいずれかの生物種に由来する酵素が挙げられる。好ましくは配列番号１５に記載されるアミノ酸配列からなるＴｈｅｒｍｏｔｈｅｌｏｍｙｃｅｓ　ｔｈｅｒｍｏｐｈｉｌｕｓ由来の酵素、配列番号１６に記載されるアミノ酸配列からなるＣｈａｅｔｏｍｉｕｍ　ｔｈｅｒｍｏｐｈｉｌｕｍ由来の酵素、および配列番号１７に記載されるアミノ酸配列からなるＣａｎｄｉｄａ　ｔｒｏｐｉｃａｌｉｓ由来の酵素が使用される（図２）。 本発明で使用される２，３－デヒドロアジピルＣｏＡレダクターゼは、例えば、Ｃａｎｄｉｄａ　ａｕｒｉｓ、Ｋｌｕｙｖｅｒｏｍｙｃｅｓ　ｍａｒｘｉａｎｕｓ、Ｐｉｃｈｉａ　ｋｕｄｒｉａｖｚｅｖｉｉ、Ｔｈｅｒｍｏｔｈｅｌｏｍｙｃｅｓ　ｔｈｅｒｍｏｐｈｉｌｕｓ、Ｔｈｅｒｍｏｔｈｉｅｌａｖｉｏｉｄｅｓ　ｔｅｒｒｅｓｔｒｉｓ、Ｃｈａｅｔｏｍｉｕｍ　ｔｈｅｒｍｏｐｈｉｌｕｍ、Ｐｏｄｏｓｐｏｒａ　ａｎｓｅｒｉｎａ、Ｐｕｒｐｕｒｅｏｃｉｌｌｉｕｍ　ｌｉｌａｃｉｎｕｍ、Ｐｙｒｅｎｏｐｈｏｒａ　ｔｅｒｅｓから選択されるいずれEnzymes derived from certain species can be mentioned. Preferably, the enzyme derived from Thermothelomyces thermophilus consisting of the amino acid sequence set forth in SEQ ID NO: 15, the enzyme derived from Chaetomium thermophilum consisting of the amino acid sequence set forth in SEQ ID NO: 16, and the Candida tropicalis consisting of the amino acid sequence set forth in SEQ ID NO: 17 Enzymes from A. are used (Fig. 2).
　図１のステップＥの変換では、アジピル－ＣｏＡがアジピン酸へと変換される。本変換を触媒し得る酵素の例としては、ＥＣ　３．１．２の群に分類されるチオエステルヒドラターゼが挙げられる。例えば、ＥＣ　３．１．２．１（アセチル－ＣｏＡヒドラターゼ）およびＥＣ　３．１．２．２０（アシル－ＣｏＡヒドラターゼ）といった群に分類される酵素は、本変換に対して活性を有し得る酵素として例示することができる。 In the conversion in step E of Figure 1, adipyl-CoA is converted to adipic acid. Examples of enzymes that can catalyze this conversion include thioester hydratases that are classified in group EC 3.1.2. For example, enzymes falling into the groups EC 3.1.2.1 (acetyl-CoA hydratase) and EC 3.1.2.20 (acyl-CoA hydratase) may have activity for this conversion. It can be exemplified as an enzyme.
　また、図１のステップＥの変換を触媒し得る別の酵素の例として、ＥＣ　２．８．３の群に分類されるＣｏＡ－トランスフェラーゼも挙げることができる。例えば、ＥＣ　２．８．３．５（３－オキソ酸　ＣｏＡ－トランスフェラーゼ）、ＥＣ　２．８．３．６（３－オキソアジピン酸　ＣｏＡ－トランスフェラーゼ）およびＥＣ　２．８．３．１８（スクシニル－ＣｏＡ：酢酸　ＣｏＡ－トランスフェラーゼ）といった群に分類される酵素は、本変換に対して活性を有し得る酵素として例示することができる。 Another example of an enzyme that can catalyze the conversion of step E in FIG. 1 is CoA-transferase, which is classified in the group EC 2.8.3. For example, EC 2.8.3.5 (3-oxoacid CoA-transferase), EC 2.8.3.6 (3-oxoadipate CoA-transferase) and EC 2.8.3.18 (succinyl- Enzymes classified into groups such as CoA:acetate (CoA-transferase) can be exemplified as enzymes that may have activity for this conversion.
　さらには、図１のステップＥの変換を触媒し得る別の酵素変換の例として、ＥＣ　２．３．１の群に分類されるアシルトランスフェラーゼによって、アジピル－ＣｏＡのアジピル基をリン酸に転移してアジピルリン酸を生成した後、ＥＣ　２．７．２の群に分類されるホスホトランスフェラーゼによる脱リン酸化を経る経路も例示することができる。例えば、アシルトランスフェラーゼとしては、ＥＣ　２．３．１．８（リン酸アセチルトランスフェラーゼ）およびＥＣ　２．３．１．１９（リン酸ブチリルトランスフェラーゼ）といった群に分類される酵素、ホスホトランスフェラーゼについては、ＥＣ　２．７．２．１（酢酸キナーゼ）およびＥＣ　２．７．２．７（ブタン酸キナーゼ）といった群に分類される酵素が、本変換に対して活性を有し得る酵素として例示することができる。 Furthermore, as another example of an enzymatic transformation that can catalyze the transformation of step E of FIG. A pathway that undergoes dephosphorylation by phosphotransferases classified in the group of EC 2.7.2 after producing adipyl phosphate can also be exemplified. For example, acyltransferases include enzymes classified into groups such as EC 2.3.1.8 (phosphate acetyltransferase) and EC 2.3.1.19 (phosphate butyryltransferase). Enzymes classified into groups such as EC 2.7.2.1 (acetate kinase) and EC 2.7.2.7 (butanoate kinase) are exemplified as enzymes that can have activity for this conversion. can be done.
　図１のステップＦの変換では、アジピル－ＣｏＡからアジピン酸セミアルデヒドへと変換される。本変換を触媒し得る酵素の例としては、ＥＣ　１．２．１の群に分類される酵素が挙げられる。例えば、ＥＣ　１．２．１．１０（アセトアルデヒドデヒドロゲナーゼ（アセチル化））、ＥＣ　１．２．１．１７（グリオキシル酸デヒドロゲナーゼ（アシル化））、ＥＣ　１．２．１．４２（ヘキサデカナールデヒドロゲナーゼ（アシル化））、ＥＣ　１．２．１．４４（シナモイル－ＣｏＡレダクターゼ（アシル化））、ＥＣ　１．２．１．７５（マロニル－ＣｏＡレダクターゼ（マロン酸セミアルデヒド形成））、ＥＣ　１．２．１．７６（コハク酸セミアルデヒドデヒドロゲナーゼ（アシル化））といった群に分類される酵素は、本変換と同様に、ＣｏＡを脱離しアルデヒドを生成する変換反応を触媒することから、本変換に対しても活性を有し得る酵素として例示することができる。本発明で使用される酵素は、本変換に対する活性を有するものであれば限定されないが、例えば、配列番号１８に記載されるアミノ酸配列からなるＣｌｏｓｔｒｉｄｉｕｍ　ｋｌｕｙｖｅｒｉ由来のｓｕｃＤが使用される（図２）。 In the conversion in step F of Figure 1, adipyl-CoA is converted to adipic acid semialdehyde. Examples of enzymes that can catalyze this conversion include enzymes classified in group EC 1.2.1. For example, EC 1.2.1.10 (acetaldehyde dehydrogenase (acetylation)), EC 1.2.1.17 (glyoxylate dehydrogenase (acylation)), EC 1.2.1.42 (hexadecanal dehydrogenase (acylation)), EC 1.2.1.44 (cinnamoyl-CoA reductase (acylation)), EC 1.2.1.75 (malonyl-CoA reductase (malonic semialdehyde formation)), EC 1. Enzymes classified into groups such as 2.1.76 (succinic semialdehyde dehydrogenase (acylation)) catalyze conversion reactions that eliminate CoA and generate aldehydes, similar to this conversion. It can also be exemplified as an enzyme that can have activity against The enzyme used in the present invention is not limited as long as it has activity for this conversion. For example, Clostridium kluyveri-derived sucD consisting of the amino acid sequence set forth in SEQ ID NO: 18 is used (Fig. 2).
　図１のステップＧ、Ｉ、Ｋ、Ｎの変換では、カルボキシル基がアルデヒドへと変換される。本変換を触媒し得る酵素としては、例えば、カルボン酸レダクターゼ（Ｃａｒｂｏｘｙｌｉｃ　Ａｃｉｄ　Ｒｅｄｕｃｔａｓｅ；ＣＡＲ）が挙げられる。例えば、ＥＣ　１．２．１．３０（カルボン酸レダクターゼ（ＮＡＤＰ＋））、ＥＣ　１．２．１．３１（Ｌ－アミノアジピン酸セミアルデヒドデヒドロゲナーゼ）、ＥＣ　１．２．１．９５（Ｌ－２－アミノアジピン酸レダクターゼ）、ＥＣ　１．２．９９．６（カルボン酸レダクターゼ）といった群に分類される酵素が、本変換と同様に、カルボン酸からアルデヒドを生成する変換反応を触媒することから、本変換に対しても活性を有し得る酵素として例示することができる。酵素の由来となる生物種の典型的な例としては、Ｎｏｃａｒｄｉａ　ｉｏｗｅｎｓｉｓ、Ｎｏｃａｒｄｉａ　ａｓｔｅｒｏｉｄｅｓ、Ｎｏｃａｒｄｉａ　ｂｒａｓｉｌｉｅｎｓｉｓ、Ｎｏｃａｒｄｉａ　ｆａｒｃｉｎｉｃａ、Ｓｅｇｎｉｌｉｐａｒｕｓ　ｒｕｇｏｓｕｓ、Ｓｅｇｎｉｌｉｐａｒｕｓ　ｒｏｔｕｎｄｕｓ、Ｔｓｕｋａｍｕｒｅｌｌａ　ｐａｕｒｏｍｅｔａｂｏｌａ、Ｍｙｃｏｂａｃｔｅｒｉｕｍ　ｍａｒｉｎｕｍ、Ｍｙｃｏｂａｃｔｅｒｉｕｍ　ｎｅｏａｕｒｕｍ、Ｍｙｃｏｂａｃｔｅｒｉｕｍ　ａｂｓｃｅｓｓｕｓ、Ｍｙｃｏｂａｃｔｅｒｉｕｍ　ａｖｉｕｍ、Ｍｙｃｏｂａｃｔｅｒｉｕｍ　ｃｈｅｌｏｎａｅ、Ｍｙｃｏｂａｃｔｅｒｉｕｍ　ｉｍｍｕｎｏｇｅｎｕｍ、Ｍｙｃｏｂａｃｔｅｒｉｕｍ　ｓｍｅｇｍａｔｉｓ、Ｓｅｒｐｕｌａ　ｌａｃｒｙｍａｎｓ、Ｈｅｔｅｒｏｂａｓｉｄｉｏｎ　ａｎｎｏｓｕｍ、Ｃｏｐｒｉｎｏｐｓｉｓ　ｃｉｎｅｒｅａ、Ａｓｐｅｒｇｉｌｌｕｓ　ｆｌａｖｕｓ、Ａｓｐｅｒｇｉｌｌｕｓ　ｔｅｒｒｅｕｓ、Ｎｅｕｒｏｓｐｏｒａ　ｃｒａｓｓａ、Ｓａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅが挙げられるが、これらに限定されない。本発明で使用される酵素は、本変換に対する活性を有するものであれば限定されないが、例えば配列番号１９に記載されるアミノ酸配列からなるＭｙｃｏｂａｃｔｅｒｉｕｍ　ａｂｓｃｅｓｓｕｓ由来の酵素ＭａＣａｒ、配列番号２０に記載されるアミノ酸配列からなるＭａＣａｒの変異体であるＭａＣａｒ（ｍ）の少なくとも一方が使用され、より好ましくは配列番号２０に記載されるアミノ酸配列からなるＭａＣａｒ（ｍ）が使用される（図２）。 In the transformations in steps G, I, K, N of Figure 1, the carboxyl group is transformed into an aldehyde. Enzymes that can catalyze this conversion include, for example, carboxylic acid reductase (CAR). For example, EC 1.2.1.30 (carboxylic acid reductase (NADP + )), EC 1.2.1.31 (L-aminoadipate semialdehyde dehydrogenase), EC 1.2.1.95 (L- 2-aminoadipate reductase) and EC 1.2.99.6 (carboxylic acid reductase) catalyzes the conversion reaction of carboxylic acid to aldehyde in the same manner as this conversion. , can be exemplified as enzymes that may also have activity for this conversion.酵素の由来となる生物種の典型的な例としては、Ｎｏｃａｒｄｉａ ｉｏｗｅｎｓｉｓ、Ｎｏｃａｒｄｉａ ａｓｔｅｒｏｉｄｅｓ、Ｎｏｃａｒｄｉａ ｂｒａｓｉｌｉｅｎｓｉｓ、Ｎｏｃａｒｄｉａ ｆａｒｃｉｎｉｃａ、Ｓｅｇｎｉｌｉｐａｒｕｓ ｒｕｇｏｓｕｓ、Ｓｅｇｎｉｌｉｐａｒｕｓ ｒｏｔｕｎｄｕｓ、Ｔｓｕｋａｍｕｒｅｌｌａ ｐａｕｒｏｍｅｔａｂｏｌａ、Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｍａｒｉｎｕｍ、Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｎｅｏａｕｒｕｍ、Ｍｙｃｏｂａｃｔｅｒｉｕｍ ａｂｓｃｅｓｓｕｓ、Ｍｙｃｏｂａｃｔｅｒｉｕｍ ａｖｉｕｍ、Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｃｈｅｌｏｎａｅ 、Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｉｍｍｕｎｏｇｅｎｕｍ、Ｍｙｃｏｂａｃｔｅｒｉｕｍ ｓｍｅｇｍａｔｉｓ、Ｓｅｒｐｕｌａ ｌａｃｒｙｍａｎｓ、Ｈｅｔｅｒｏｂａｓｉｄｉｏｎ ａｎｎｏｓｕｍ、Ｃｏｐｒｉｎｏｐｓｉｓ ｃｉｎｅｒｅａ、Ａｓｐｅｒｇｉｌｌｕｓ ｆｌａｖｕｓ、Ａｓｐｅｒｇｉｌｌｕｓ ｔｅｒｒｅｕｓ、Ｎｅｕｒｏｓｐｏｒａ ｃｒａｓｓａ、Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅが挙げられるが、これらに限定されない。 The enzyme used in the present invention is not limited as long as it has activity for this conversion. At least one of MaCar(m), which is a mutant of MaCar consisting of a sequence, is used, and more preferably MaCar(m) consisting of the amino acid sequence set forth in SEQ ID NO: 20 is used (Fig. 2).
　また、カルボン酸レダクターゼは、ホスホパンテテイニル化されることにより活性型のホロ酵素に変換され得る（Ｖｅｎｋｉｔａｓｕｂｒａｍａｎｉａｎ　ｅｔ　ａｌ．，　Ｊｏｕｒｎａｌ　ｏｆ　Ｂｉｏｌｏｇｉｃａｌ　Ｃｈｅｍｉｓｔｒｙ，Ｖｏｌ．２８２，Ｎｏ．１，４７８－４８５（２００７））。ホスホパンテテイニル化はホスホパンテテイニル基転移酵素（Ｐｈｏｓｐｈｏｐａｎｔｅｔｈｅｉｎｙｌ　Ｔｒａｎｓｆｅｒａｓｅ；ＰＴ）により触媒される。本反応を触媒し得る酵素としては、例えば、ＥＣ　２．７．８．７に分類される酵素が挙げられる。したがって、本発明の微生物は更に、ホスホパンテテイニル基転移酵素の活性が増大するように改変されていて良い。ホスホパンテテイニル基転移酵素の活性を増大する方法としては、外来のホスホパンテテイニル基転移酵素遺伝子を導入する方法、および、内因性のホスホパンテテイニル基転移酵素遺伝子の発現を強化する方法が挙げられるが、これらに限定されない。本発明で使用される酵素は、ホスホパンテテイニル基転移活性を有する限り、これらに限定されないが、典型的な酵素としては、例えば、大腸菌のＥｎｔＤ、Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓのＳｆｐ、Ｎｏｃａｒｄｉａ　ｉｏｗｅｎｓｉｓのＮｐｔ（Ｖｅｎｋｉｔａｓｕｂｒａｍａｎｉａｎ　ｅｔ　ａｌ．，　Ｊｏｕｒｎａｌ　ｏｆ　Ｂｉｏｌｏｇｉｃａｌ　Ｃｈｅｍｉｓｔｒｙ，Ｖｏｌ．２８２，Ｎｏ．１，４７８－４８５（２００７））、およびＳａｃｃｈａｒｏｍｙｃｅｓ　ｃｅｒｅｖｉｓｉａｅのＬｙｓ５（Ｅｈｍａｎｎ　ｅｔ　ａｌ．，　Ｂｉｏｃｈｅｍｉｓｔｒｙ　３８．１９　（１９９９）：　６１７１－６１７７．）が挙げられる。本発明で使用される酵素は、本変換に対する活性を有するものであれば限定されないが、例えば、配列番号２１に記載されるアミノ酸配列からなるＮｏｃａｒｄｉａ　ｉｏｗｅｎｓｉｓ由来のＮｐｔが使用される（図２）。 In addition, carboxylic acid reductase can be converted to an active holoenzyme by phosphopantetheinylation (Venkitasubramanian et al., Journal of Biological Chemistry, Vol. 282, No. 1, 478-485 (2007) ). Phosphopantetheinylation is catalyzed by phosphopantetheinyl transferase (PT). Enzymes that can catalyze this reaction include, for example, enzymes classified under EC 2.7.8.7. Accordingly, the microorganism of the invention may be further modified to increase the activity of phosphopantetheinyltransferase. Methods of increasing the activity of phosphopantetheinyl transferase include a method of introducing an exogenous phosphopantetheinyl transferase gene and a method of enhancing expression of an endogenous phosphopantetheinyl transferase gene. include, but are not limited to. Enzymes used in the present invention are not limited to these as long as they have phosphopantetheinyl group transfer activity. et al., Journal of Biological Chemistry, Vol. 282, No. 1, 478-485 (2007)) and Lys5 of Saccharomyces cerevisiae (Ehmann et al., Biochemistry 38.19 (1999): 16171-6). mentioned. The enzyme used in the present invention is not limited as long as it has activity for this conversion. For example, Npt derived from Nocardia iowensis consisting of the amino acid sequence set forth in SEQ ID NO: 21 is used (Fig. 2).
　図１のステップＪ、Ｍ、Ｐ、Ｒの変換は、アミノ基転移反応である。この変換を触媒し得る酵素の例としては、ＥＣ　２．６．１の群に分類されるトランスアミナーゼ（アミノトランスフェラーゼ）が挙げられる。例えば、ＥＣ　２．６．１．１９（４－アミノブタン酸－２－オキソグルタル酸トランスアミナーゼ）およびＥＣ　２．６．１．２９（ジアミントランスアミナーゼ）、ＥＣ　２．６．１．４８（５－アミノ吉草酸トランスアミナーゼ）といった群に分類される酵素は、本変換に対しても活性を有し得る酵素として例示することができる。本発明で使用される酵素は、各ステップの変換活性を有するものであれば、特に限定されないが、例えば、カダベリンおよびスペルミジンをアミノ基転移することが報告されている大腸菌のプトレシンアミノトランスフェラーゼであるＹｇｊＧ（Ｓａｍｓｏｎｏｖａ．，　ｅｔ　ａｌ．，　ＢＭＣ　ｍｉｃｒｏｂｉｏｌｏｇｙ　３．１　（２００３）：　２．）、シュードモナス属のプトレシンアミノトランスフェラーゼであるＳｐｕＣ（Ｌｕ　ｅｔ　ａｌ．，Ｊｏｕｒｎａｌ　ｏｆ　ｂａｃｔｅｒｉｏｌｏｇｙ　１８４．１４　（２００２）：　３７６５－３７７３．、Ｇａｌｍａｎ　ｅｔ　ａｌ．，Ｇｒｅｅｎ　Ｃｈｅｍｉｓｔｒｙ　１９．２　（２０１７）：　３６１－３６６．）、大腸菌のＧＡＢＡアミノトランスフェラーゼＧａｂＴ、ならびに、ＰｕｕＥが使用されてもよい。さらには、Ｒｕｅｇｅｒｉａ　ｐｏｍｅｒｏｙｉ、Ｃｈｒｏｍｏｂａｃｔｅｒｉｕｍ　ｖｉｏｌａｃｅｕｍ、Ａｒｔｈｒｏｂａｃｔｅｒ　ｃｉｔｒｅｕｓ、Ｓｐｈａｅｒｏｂａｃｔｅｒ　ｔｈｅｒｍｏｐｈｉｌｕｓ、Ａｓｐｅｒｇｉｌｌｕｓ　ｆｉｓｃｈｅｒｉ、Ｖｉｂｒｉｏ　ｆｌｕｖｉａｌｉｓ、Ａｇｒｏｂａｃｔｅｒｉｕｍ　ｔｕｍｅｆａｃｉｅｎｓ、Ｍｅｓｏｒｈｉｚｏｂｉｕｍ　ｌｏｔｉ等の生物種由来のω－トランスアミナーゼも、１，８－ジアミノオクタンおおよび１，１０－ジアミノデカンなどのジアミン化合物へのアミノ基転移活性を有することが報告されており、本発明で使用されてもよい（Ｓｕｎｇ　ｅｔ　ａｌ．，　Ｇｒｅｅｎ　Ｃｈｅｍｉｓｔｒｙ　２０．２０　（２０１８）：　４５９１－４５９５．、Ｓａｔｔｌｅｒ　ｅｔ　ａｌ．，　Ａｎｇｅｗａｎｄｔｅ　Ｃｈｅｍｉｅ　１２４．３６　（２０１２）：　９２９０－９２９３．）。本発明で使用される酵素は、本変換に対する活性を有するものであれば限定されないが、例えば、配列番号２２に記載されるアミノ酸配列からなる大腸菌由来の酵素ＹｇｊＧが使用されてもよい（図２）。典型的なアミノ基供与体としては、Ｌ－グルタミン酸、Ｌ－アラニン、グリシンが挙げられるが、これらには限定されない。 The transformations in steps J, M, P, and R in Figure 1 are transamination reactions. Examples of enzymes that can catalyze this conversion include the transaminases (aminotransferases) that fall into the group of EC 2.6.1. For example, EC 2.6.1.19 (4-aminobutanoic acid-2-oxoglutarate transaminase) and EC 2.6.1.29 (diamine transaminase), EC 2.6.1.48 (5-aminovalerate Enzymes classified into groups such as transaminase) can be exemplified as enzymes that may also have activity for this conversion. The enzyme used in the present invention is not particularly limited as long as it has the conversion activity of each step. (Samsonova., et al., BMC Microbiology 3.1 (2003): 2.), Pseudomonas putrescine aminotransferase SpuC (Lu et al., Journal of bacteria 184.14 (2002): 3765-3773. , Galman et al., Green Chemistry 19.2 (2017): 361-366.), the E. coli GABA aminotransferase GabT, and PuuE may be used.さらには、Ｒｕｅｇｅｒｉａ　ｐｏｍｅｒｏｙｉ、Ｃｈｒｏｍｏｂａｃｔｅｒｉｕｍ　ｖｉｏｌａｃｅｕｍ、Ａｒｔｈｒｏｂａｃｔｅｒ　ｃｉｔｒｅｕｓ、Ｓｐｈａｅｒｏｂａｃｔｅｒ　ｔｈｅｒｍｏｐｈｉｌｕｓ、Ａｓｐｅｒｇｉｌｌｕｓ　ｆｉｓｃｈｅｒｉ、Ｖｉｂｒｉｏ　ｆｌｕｖｉａｌｉｓ、Ａｇｒｏｂａｃｔｅｒｉｕｍ　ｔｕｍｅｆａｃｉｅｎｓ、Ｍｅｓｏｒｈｉｚｏｂｉｕｍ　ｌｏｔｉ等の生物種由来のω－トランスアミナーゼも、１，８－ジアミノオクタンおおよび１，１０－ジアミノIt is reported to have transamination activity to diamine compounds such as decane and may be used in the present invention (Sung et al., Green Chemistry 20.20 (2018): 4591-4595., Sattler et al., Green Chemistry 20.20 (2018): 4591-4595. al., Angewandte Chemie 124.36 (2012): 9290-9293.). The enzyme used in the present invention is not limited as long as it has activity for this conversion. For example, an E. coli-derived enzyme YgjG consisting of the amino acid sequence set forth in SEQ ID NO: 22 may be used (Fig. 2 ). Typical amino group donors include, but are not limited to, L-glutamic acid, L-alanine, glycine.
　本発明に利用できる上記の酵素をコードする遺伝子は、例示された微生物以外に由来するものであっても、または人工的に合成したものであってもよく、前記宿主微生物細胞内で実質的な酵素活性を発現できるものであればよい。 Genes encoding the above enzymes that can be used in the present invention may be derived from microorganisms other than the exemplified microorganisms, or may be artificially synthesized, and substantially Any material can be used as long as it can express enzymatic activity.
　また、本発明の目的に利用できる上記酵素遺伝子は、前記宿主微生物細胞内で実質的な酵素活性を発現できるものであれば、自然界で発生し得るすべての変異、ならびに、人工的に導入された変異及び修飾を有していてもよい。例えば、特定のアミノ酸をコードする種々のコドンには余分のコドンが存在することが知られている。そのため本発明においても同一のアミノ酸に最終的に翻訳されることになる代替コドンを利用してよい。つまり、遺伝子コードは縮重しているので、ある特定のアミノ酸をコードするのに複数のコドンを使用でき、そのためアミノ酸配列は任意の１セットの類似のＤＮＡオリゴヌクレオチドでコードされ得る。そのセットの唯一のメンバーだけが天然型酵素の遺伝子配列に同一であるが、ミスマッチのあるＤＮＡオリゴヌクレオチドでさえ適切な緊縮条件下（例えば、３ｘＳＳＣ、６８℃でハイブリダイズし、２ｘＳＳＣ、０．１％ＳＤＳ及び６８℃で洗浄）で天然型配列にハイブリダイズでき、天然型配列をコードするＤＮＡを同定、単離でき、更にそのような遺伝子も本発明において利用できる。特に、ほとんどの生物は特定のコドン（最適コドン）のサブセットを優先的に用いることが知られているので（Ｇｅｎｅ、Ｖｏｌ．１０５、ｐｐ．６１－７２、１９９１等）、宿主微生物に応じて「コドン最適化」を行うことは本発明においても有用であり得る。 In addition, the enzyme gene that can be used for the purpose of the present invention includes all mutations that can occur in nature, as well as artificially introduced enzyme genes, as long as they can express substantial enzyme activity in the host microbial cells. It may have variations and modifications. For example, it is known that there are extra codons for various codons that code for specific amino acids. Therefore, also in the present invention, alternative codons that are ultimately translated into the same amino acid may be used. That is, because the genetic code is degenerate, multiple codons can be used to encode a particular amino acid, such that an amino acid sequence can be encoded by any set of similar DNA oligonucleotides. Only one member of the set is identical to the gene sequence of the native enzyme, but even mismatched DNA oligonucleotides hybridize under appropriate stringency conditions (e.g., 3xSSC, 68°C, 2xSSC, 0.1 % SDS and washed at 68° C.), DNA encoding the native sequence can be identified and isolated, and such genes can also be used in the present invention. In particular, since it is known that most organisms preferentially use a subset of specific codons (optimal codons) (Gene, Vol.105, pp.61-72, 1991, etc.), depending on the host microorganism " Performing "codon optimization" may also be useful in the present invention.
　ジアミンが１，５－ペンタメチレンジアミンである場合、好ましい態様において、組換え微生物は、配列番号２または４に示す塩基配列と、８５、９０、９２、９５、９８または９９％以上の相同性、好ましくは９２、９５、９８または９９％以上の相同性を有する塩基配列を含むか、あるいは、組換え微生物は、配列番号１または３に示すアミノ酸配列をコードする塩基配列と８５、９０、９２、９５、９８または９９％以上の相同性、好ましくは９２、９５、９８または９９％以上の相同性を有する塩基配列を含む。 When the diamine is 1,5-pentamethylenediamine, in a preferred embodiment, the recombinant microorganism has 85, 90, 92, 95, 98 or 99% or more homology with the nucleotide sequence shown in SEQ ID NO: 2 or 4, Preferably, the recombinant microorganism contains a nucleotide sequence having 92, 95, 98 or 99% or more homology, or the recombinant microorganism has a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 3 and 85, 90, 92, It includes nucleotide sequences with 95, 98 or 99% or more homology, preferably 92, 95, 98 or 99% or more homology.
　例えば、上記のジアミン合成酵素遺伝子が「発現カセット」として宿主微生物細胞内に導入されることで、安定的で高レベルの酵素活性を得ることができる。好ましくは、「発現カセット」の遺伝子配列が宿主微生物のゲノム配列に挿入されることで、より安定的で高レベルの酵素活性を得ることができる。 For example, by introducing the above-mentioned diamine synthase gene as an "expression cassette" into host microbial cells, stable and high-level enzymatic activity can be obtained. Preferably, the gene sequence of the "expression cassette" is inserted into the genome sequence of the host microorganism to obtain more stable and high-level enzymatic activity.
　ここで、本明細書において、「発現カセット」とは、発現対象の核酸または発現対象の遺伝子に機能的に結合された転写および翻訳をレギュレートする核酸配列を含むヌクレオチドを意味する。典型的に、本発明の発現カセットは、コード配列から５’上流にプロモーター配列、３’下流にターミネーター配列、場合により更なる通常の調節エレメントを機能的に結合された状態で含み、そのような場合に、発現対象の核酸または発現対象の遺伝子が宿主微生物に導入される。 As used herein, the term "expression cassette" means a nucleotide containing a nucleic acid sequence that regulates transcription and translation, functionally linked to a nucleic acid to be expressed or a gene to be expressed. Typically, an expression cassette of the invention comprises a promoter sequence 5′ upstream from the coding sequence, a terminator sequence 3′ downstream, and optionally further conventional regulatory elements operably linked, such that In some cases, the nucleic acid to be expressed or the gene to be expressed is introduced into the host microorganism.
　また、プロモーターとは、構成発現型プロモーターであるか誘導発現型プロモーターであるかに拘わらず、ＲＮＡポリメラーゼをＤＮＡに結合させ、ＲＮＡ合成を開始させるＤＮＡ配列と定義される。強いプロモーターとはｍＲＮＡ合成を高頻度で開始させるプロモーターであり、本発明においても好適に使用される。例えば、バチルス・シュードフィラマスではＳ－Ｌａｙｅｒタンパク質の合成酵素、シグマ因子（例えばｒｐｏＤなど）、解糖系酵素（例えば、グリセルアルデヒド－３－リン酸脱水素酵素）、乳酸脱水素酵素、グルタミン酸デカルボキシラーゼＡに対するプロモーター領域等が利用可能である。 A promoter is defined as a DNA sequence that causes RNA polymerase to bind to DNA and initiate RNA synthesis, regardless of whether it is a constitutive promoter or an inducible promoter. A strong promoter is a promoter that initiates mRNA synthesis at a high frequency, and is also preferably used in the present invention. For example, in Bacillus pseudofilamus, S-Layer protein synthetase, sigma factor (eg, rpoD), glycolytic enzyme (eg, glyceraldehyde-3-phosphate dehydrogenase), lactate dehydrogenase, glutamic acid A promoter region or the like for decarboxylase A is available.
　上記で説明した発現カセットは、例えば、プラスミド、ファージ、トランスポゾン、ＩＳエレメント、ファスミド、コスミド、または線状もしくは環状のＤＮＡ等から成るベクターに組み入れて、宿主微生物中に導入される。本発明ではプラスミドおよびファージが好ましい。これらのベクターは、宿主微生物中で自律複製されるものでもよいし、また染色体に挿入され複製されてもよい。好適なプラスミドは、例えば、バチルス属などの桿菌ではｐＵＢ１１０、ｐＣ１９４およびｐＢＤ２１４などが挙げられる。 The expression cassette described above is incorporated into a vector consisting of, for example, a plasmid, phage, transposon, IS element, phasmid, cosmid, or linear or circular DNA, and introduced into a host microorganism. Plasmids and phages are preferred in the present invention. These vectors may replicate autonomously in host microorganisms, or may replicate by being inserted into chromosomes. Suitable plasmids include, for example, pUB110, pC194 and pBD214 for bacilli such as Bacillus.
　上記で説明した発現カセットは、プラスミドおよびファージに比べ、染色体に挿入されていることが好ましい。宿主微生物内においてプラスミドを保持させるためには何らかの選択圧が必要であり、一般的にはプラスミドが持つ抗生物質耐性マーカーに対応する抗生物質の培地への添加が必要になる。また、選択圧があったとしても、プラスミド上で発現される遺伝子が宿主微生物にとって不必要あるいは生育に負荷となる場合に、宿主微生物が内在的に保持する酵素の働きにより該当遺伝子上に変異が導入される、あるいは削除されるなどの可能性があり、物質の安定生産が困難となる場合が多い。 The expression cassette described above is preferably inserted into the chromosome compared to plasmids and phages. Some selective pressure is required to maintain the plasmid within the host microorganism, generally requiring the addition of an antibiotic to the culture medium corresponding to the antibiotic resistance marker carried by the plasmid. In addition, even if there is selective pressure, if the gene expressed on the plasmid is unnecessary or burdens the growth of the host microorganism, the function of the enzyme endogenously retained by the host microorganism will cause mutation on the gene. There is a possibility that they may be introduced or deleted, and in many cases stable production of substances becomes difficult.
　使用可能なプラスミド等としては、上記の他、”Ｃｌｏｎｉｎｇ　Ｖｅｃｔｏｒｓ”、Ｅｌｓｅｖｉｅｒ、１９８５に記載されているものが挙げられる。ベクターへの発現カセットの導入は、ＰＣＲによる断片増幅、適当な制限酵素による切り出し、クローニング、及び種々のライゲーションを含む慣用の方法によって可能である。 In addition to the above, usable plasmids and the like include those described in "Cloning Vectors", Elsevier, 1985. Introduction of an expression cassette into a vector can be performed by conventional methods including fragment amplification by PCR, excision with appropriate restriction enzymes, cloning, and various types of ligation.
　上記のようにして本発明の発現カセットを有するベクターが構築された後、該ベクターを宿主微生物に導入する際に適用できる手法として、例えば、接合伝達、共沈、プロトプラスト融合、エレクトロポレーション、レトロウイルストランスフェクションなどの慣用のクローニング法およびトランスフェクション法が使用される。それらの例は、「分子生物学の最新プロトコル（Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ）」、Ｆ．　Ａｕｓｕｂｅｌら、Ｐｕｂｌ．Ｗｉｌｅｙ　Ｉｎｔｅｒｓｃｉｅｎｃｅ、Ｎｅｗ　Ｙｏｒｋ、１９９７、またはＳａｍｂｒｏｏｋら、「分子クローニング：実験室マニュアル」、第２版、Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ、Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ、Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ、ＮＹ、１９８９に記載されている。 After the vector having the expression cassette of the present invention is constructed as described above, techniques that can be applied when introducing the vector into a host microorganism include, for example, conjugative transfer, coprecipitation, protoplast fusion, electroporation, retro Conventional cloning and transfection methods are used, such as viral transfection. Examples of these can be found in "Current Protocols in Molecular Biology", F.　Ausubel et al., Publ. Wiley Interscience, New York, 1997, or Sambrook et al., "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
　さらに、プラスミドベクターの配列に温度感受性の複製起点配列、ネガティブセレクション遺伝子、宿主微生物のゲノム配列の編集したい領域の両側の相同配列を含ませることで、宿主微生物のゲノム配列に外来遺伝子を導入すること、または不要な遺伝子を除去することができる。好ましくは温度感受性の複製起点配列がｐＥ１９４ｔｓベクターで使用されている複製起点であり、ネガティブセレクション遺伝子がｓａｃＢ遺伝子または変異ｐｈｅＳ遺伝子である。 Furthermore, by including a temperature-sensitive replication origin sequence, a negative selection gene, and homologous sequences on both sides of the region to be edited in the genome sequence of the host microorganism in the sequence of the plasmid vector, a foreign gene can be introduced into the genome sequence of the host microorganism. , or unwanted genes can be removed. Preferably, the temperature sensitive origin of replication sequence is the origin of replication used in the pE194ts vector and the negative selection gene is the sacB gene or the mutated pheS gene.
　上記で説明したベクターが導入された宿主微生物を培養する過程で、温度変化と培地組成変化によりゲノム配列が改変された編集体を選択することができる。編集体を選択する時の条件としては、培養温度が３７～４３℃以上であること、培地に１０％以上のスクロースが含まれていること、あるいは培地に１ｍＭ以上の４－クロロフェニルアラニンが含まれていることが好ましい。 In the process of culturing the host microorganism into which the vector described above has been introduced, it is possible to select an editor whose genome sequence has been modified by temperature change and medium composition change. The conditions for selecting editorial bodies are that the culture temperature is 37 to 43° C. or higher, that the medium contains 10% or higher sucrose, or that the medium contains 1 mM or higher 4-chlorophenylalanine. preferably.
　生産させるジアミンが１，５－ペンタメチレンジアミンである場合、好ましい一態様において、宿主微生物には、リジン生産能を向上させるために、突然変異操作または遺伝子組換え操作による改変がさらに行われており、すなわち、得られる組換え微生物は、リジン生産能を向上させるための突然変異操作または遺伝子組換え操作による改変をさらに含んでいる。当該突然変異操作または前記遺伝子組換え操作は、例えば、アスパルトキナーゼＩＩＩ（ＥＣ　２．７．２．４）及び４－ヒドロキシ－テトラヒドロジピコリン酸シンターゼ（ＥＣ　４．３．３．７）の少なくとも一方に対する、フィードバック阻害を解除する操作である。 When the diamine to be produced is 1,5-pentamethylenediamine, in a preferred embodiment, the host microorganism is further modified by mutation or genetic recombination to improve lysine-producing ability. That is, the resulting recombinant microorganism further contains modification by mutagenesis or gene recombination to improve lysine-producing ability. The mutation manipulation or the genetic recombination manipulation is, for example, at least one of aspartokinase III (EC 2.7.2.4) and 4-hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7) This is an operation to release the feedback inhibition for
　アスパルトキナーゼＩＩＩは、アスパラギン酸およびアデノシン三リン酸（ＡＴＰ）を４－ホスホアスパラギン酸とアデノシン二リン酸に変換する反応を触媒する酵素である。本酵素はＬ－リジンによってフィードバック阻害されることが一般的に認知されている。フィードバック阻害を受けないよう改変された変異酵素では、タンパク構造が変化しＬ－リジンが結合せず、リジン存在下でも酵素活性を持つことから、本酵素を発現する微生物はリジンを高生産することが知られている。以下、有効な変異例としてＥｓｃｈｅｒｉｃｉａ　ｃｏｌｉ由来のアスパルトキナーゼＩＩＩ（ｌｙｓＣ）を例として説明するが、本発明に用いる遺伝子はこれに限定されるものではない。Ｌ－リジンによるフィードバック阻害を受けない変異型のｌｙｓＣとしては、そのアミノ酸配列において３５２番目のトレオニン残基がイソロイシン残基に置換されたもの、２５３番目のトレオニン残基がアルギニン残基に置換されたものなどが挙げられるが、これらに限定されない。 Aspartokinase III is an enzyme that catalyzes the reaction that converts aspartic acid and adenosine triphosphate (ATP) into 4-phosphoaspartic acid and adenosine diphosphate. It is generally recognized that this enzyme is feedback-inhibited by L-lysine. A mutant enzyme that has been modified so as not to be subject to feedback inhibition changes its protein structure so that L-lysine does not bind, and it retains enzymatic activity even in the presence of lysine. It has been known. Aspartokinase III (lysC) derived from Eschericia coli will be described below as an example of an effective mutation, but the gene used in the present invention is not limited to this. The mutant lysC that is not subject to feedback inhibition by L-lysine has an amino acid sequence in which the 352nd threonine residue is substituted with an isoleucine residue, and the 253rd threonine residue is substituted with an arginine residue. and the like, but are not limited to these.
　４－ヒドロキシ－テトラヒドロジピコリン酸シンターゼは、ピルビン酸およびアスパラギン酸セミアルデヒドを（２Ｓ，４Ｓ）－４－ヒドロキシ－２，３，４，５－テトラヒドロ－（２Ｓ）－ジピコリン酸と水に変換する反応を触媒する酵素である。本酵素はＬ－リジンによってフィードバック阻害されることが一般的に認知されている。フィードバック阻害を受けないよう改変された変異酵素ではタンパク構造が変化しＬ－リジンが結合せず、リジン存在下でも酵素活性を持つことから、本酵素を発現する微生物はリジンを高生産することが知られている。以下、有効な変異例としてＥｓｃｈｅｒｉｃｉａ　ｃｏｌｉ由来の４－ヒドロキシ－テトラヒドロジピコリン酸シンターゼ（ｄａｐＡ）を例として説明するが、本発明に用いる遺伝子はこれに限定されるものではない。Ｌ－リジンによるフィードバック阻害を受けない変異型のｄａｐＡとしては、そのアミノ酸配列において８１番目のアラニン残基がバリン残基に置換されたもの、８４番目のグルタミン酸残基がトレオニン残基に置換されたもの、１１８番目のヒスチジン残基がアルギニン残基またはチロシン残基に置換されたものなどが挙げられるが、これらに限定されない。 4-Hydroxy-tetrahydrodipicolinate synthase converts pyruvate and aspartate semialdehyde to (2S,4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinate and water is an enzyme that catalyzes It is generally recognized that this enzyme is feedback-inhibited by L-lysine. A mutant enzyme that has been modified so that it is not subject to feedback inhibition changes its protein structure, does not bind L-lysine, and retains enzymatic activity even in the presence of lysine. Are known. As an example of effective mutation, 4-hydroxy-tetrahydrodipicolinate synthase (dapA) derived from Eschericia coli will be described below, but the gene used in the present invention is not limited to this. Mutant dapA that is not subject to feedback inhibition by L-lysine has an amino acid sequence in which the 81st alanine residue is replaced with a valine residue, and the 84th glutamic acid residue is replaced with a threonine residue. and those in which the histidine residue at position 118 is replaced with an arginine residue or a tyrosine residue, but are not limited to these.
　上記のようにして得られる形質転換体またはゲノム編集体は、ジアミン生産のために、前記形質転換体の生育及び／または維持に適した条件下で培養及び維持される。例えば、１，５－ペンタンジアミンが生産される場合、外来Ｌ－リジンデカルボキシラーゼ遺伝子の発現カセットを有するベクター（各々の発現カセットは、別個のまたは同じベクター上に配置されてよい。）により形質転換された形質転換体、または外来Ｌ－リジンデカルボキシラーゼ遺伝子の発現カセットが宿主微生物のゲノム配列に組み込まれたゲノム編集体が、１，５－ペンタンジアミン生産のために、前記形質転換体の生育及び／または維持に適した条件下で培養及び維持される。各種の宿主微生物細胞に由来する形質転換体のための好適な培地組成、培養条件、培養時間は当業者により容易に設定できる。 The transformant or genome editor obtained as described above is cultured and maintained under conditions suitable for the growth and/or maintenance of the transformant for diamine production. For example, when 1,5-pentanediamine is to be produced, transformation with a vector having an expression cassette of an exogenous L-lysine decarboxylase gene (each expression cassette may be placed on a separate vector or on the same vector). The transformed transformant or the genome editor in which the expression cassette of the exogenous L-lysine decarboxylase gene is integrated into the genome sequence of the host microorganism is used for 1,5-pentanediamine production by growing the transformant and /or cultured and maintained under conditions suitable for maintenance. Suitable medium composition, culture conditions and culture time for transformants derived from various host microbial cells can be easily determined by those skilled in the art.
　培地は、１つ以上の炭素源、窒素源、無機塩、ビタミン、及び場合により微量元素ないしビタミン等の微量成分を含む天然、半合成、合成培地であってよい。しかし、使用する培地は、培養すべき形質転換体の栄養要求を適切に満たさなければならないことは言うまでもない。 The medium may be a natural, semi-synthetic, or synthetic medium containing one or more carbon sources, nitrogen sources, inorganic salts, vitamins, and optionally trace elements or trace components such as vitamins. However, it goes without saying that the medium used must adequately meet the nutritional requirements of the transformant to be cultured.
　炭素源としては、Ｄ－グルコース、スクロース、ラクトース、フルクトース、マルトース、オリゴ糖、多糖、でんぷん、セルロース、米ぬか、廃糖蜜、油脂（例えば大豆油、ヒマワリ油、ピーナッツ油、ヤシ油など）、脂肪酸（例えばパルミチン酸、リノール酸、リノレン酸など）、アルコール（例えばグリセロール、エタノールなど）、有機酸（例えば酢酸、乳酸、コハク酸など）が挙げられる。更にＤ－グルコースを含有するバイオマスであり得る。好適なバイオマスとしては、トウモロコシ分解液およびセルロース分解液が例示される。さらにその他の炭素源として、糖、二酸化炭素、合成ガス、メタノール、及びアミノ酸等も挙げられる。これらの炭素源は、個別にあるいは混合物として使用することが出来る。 Carbon sources include D-glucose, sucrose, lactose, fructose, maltose, oligosaccharides, polysaccharides, starch, cellulose, rice bran, blackstrap molasses, fats and oils (such as soybean oil, sunflower oil, peanut oil, coconut oil, etc.), fatty acids ( palmitic acid, linoleic acid, linolenic acid, etc.), alcohols (eg, glycerol, ethanol, etc.), organic acids (eg, acetic acid, lactic acid, succinic acid, etc.). It can also be a biomass containing D-glucose. Suitable biomass includes corn hydrolyzate and cellulose hydrolyzate. Still other carbon sources include sugars, carbon dioxide, syngas, methanol, amino acids, and the like. These carbon sources can be used individually or as a mixture.
　バイオマス由来の原料を用いた場合、製品であるジアミンは、ＩＳＯ１６６２０－２またはＡＳＴＭ　Ｄ６８６６に規定されるＣａｒｂｏｎ－１４（放射性炭素）分析に基づくバイオベース炭素含有率の測定により、例えば石油、天然ガス、石炭などを由来とする合成原料と明確に区別することができる。 When biomass-derived raw materials are used, the product diamine can be obtained by measuring biobased carbon content based on Carbon-14 (radiocarbon) analysis specified in ISO 16620-2 or ASTM D6866, such as petroleum, natural gas, It can be clearly distinguished from synthetic raw materials derived from coal and the like.
　窒素源としては、含窒素有機化合物（例えば、ペプトン、酵母抽出物、肉抽出物、麦芽抽出物、コーンスティープリカー、大豆粉および尿素など）、または無機化合物（例えば、硫酸アンモニウム、塩化アンモニウム、リン酸アンモニウム、炭酸アンモニウム、硝酸ナトリウム、硝酸アンモニウムなど）が挙げられる。これらの窒素源は、個別にあるいは混合物として使用することが出来る。 Nitrogen sources include nitrogen-containing organic compounds (e.g. peptones, yeast extracts, meat extracts, malt extracts, corn steep liquor, soy flour and urea) or inorganic compounds (e.g. ammonium sulfate, ammonium chloride, phosphate ammonium, ammonium carbonate, sodium nitrate, ammonium nitrate, etc.). These nitrogen sources can be used individually or as a mixture.
　また、培地は、形質転換体が有用な付加的形質を発現する場合、例えば抗生物質への耐性マーカーを有する場合、対応する抗生物質を含んでいてよい。それにより、発酵中の雑菌による汚染リスクが低減される。抗生物質としては、アンピシリン、カナマイシン、クロラムフェニコール、テトラサイクリン、エリスロマイシン、ストレプトマイシン、スペクチノマイシンなどが挙げられるが、これらに限定されない。 The medium may also contain a corresponding antibiotic if the transformant expresses useful additional traits, for example, has a marker for resistance to an antibiotic. This reduces the risk of contamination by germs during fermentation. Antibiotics include, but are not limited to ampicillin, kanamycin, chloramphenicol, tetracycline, erythromycin, streptomycin, spectinomycin, and the like.
　上記のセルロースおよび多糖類などの炭素源を宿主微生物が資化できない場合は、当該宿主微生物に外来遺伝子を導入するなどの公知の遺伝子工学的手法を施すことで、これら炭素源を使用したジアミン生産に適応させることができる。外来遺伝子としては、例えば、セルラーゼ遺伝子およびアミラーゼ遺伝子などを挙げることができる。 If the host microorganism cannot assimilate the above carbon sources such as cellulose and polysaccharides, diamine production using these carbon sources by applying known genetic engineering techniques such as introducing exogenous genes into the host microorganism. can be adapted to Exogenous genes include, for example, cellulase genes and amylase genes.
　培養は、バッチ式であっても連続式であってもよい。また、いずれの場合にも、培養の適切な時点で追加の前記炭素源等を補給する形式であってもよい。更に、培養は、好適な温度、酸素濃度、ｐＨ等を維持しながら継続されるべきである。一般的な微生物宿主細胞に由来する形質転換体の好適な培養温度は、通常１５℃～５０℃、好ましくは２５℃～３７℃の範囲である。宿主微生物が好気性の場合、発酵中の適切な酸素濃度を確保するために振盪（フラスコ培養等）、攪拌／通気（ジャー・ファーメンター培養等）を行う必要がある。それらの培養条件は、当業者にとって容易に設定可能である。 Cultivation may be batch or continuous. Moreover, in any case, it may be possible to replenish the additional carbon source or the like at an appropriate point in the culture. Furthermore, the culture should be continued while maintaining suitable temperature, oxygen concentration, pH and the like. Suitable culture temperatures for transformants derived from common microbial host cells are usually in the range of 15°C to 50°C, preferably 25°C to 37°C. If the host microorganism is aerobic, shaking (flask culture, etc.) and agitation/aeration (jar fermenter culture, etc.) should be performed to ensure an appropriate oxygen concentration during fermentation. Those culture conditions can be easily set by those skilled in the art.
　また、本発明の別の実施形態は、先述の組換え微生物を用いてジアミンを製造する方法に関する。ジアミンを製造する方法には、例えば、以下の工程が含まれる。 In addition, another embodiment of the present invention relates to a method for producing diamine using the aforementioned recombinant microorganism. A method for producing a diamine includes, for example, the following steps.
　（ａ）培養工程
　ジアミンの製造方法には、先述の実施形態にかかる組換え微生物を培養する培養工程が含まれる。当該工程における培養によって、菌体を含む培養液が得られる。当該培養工程では、無機塩を含有する培養液で前記組換え微生物を培養してもよい。無機塩とは金属元素の塩酸塩、硫酸塩、燐酸塩、炭酸塩、フッ化水素酸塩などであり、例えば、塩化ナトリウム、塩化リチウム、硫酸ナトリウム、硫酸カリウム、硫酸マグネシウム、及び炭酸ナトリウムなどが挙げられるが、中でも硫酸ナトリウム及び炭酸ナトリウムが好ましい。いかなる理論によっても限定することを意図しないが、無機塩を水溶液に加えるとその強い水和力によって水分子を水和水として固定するため、ジアミン類の水和に必要な水分子の量が減少し、相分離が生ずると考えられる。無機塩の塩析の強さを示す指標にホフマイスター系列があるが、ホフマイスター系列に示される、塩析を強く引き起こすアニオンとカチオン（特に金属イオン）との組み合わせからなる塩が好ましい。無機塩を含有する培養液中で培養することにより、ジアミン類が生産されるが、高濃度の無機塩存在下では培養液からジアミン水溶液の相分離が生ずる。故に無機塩を培養液中に含ませることによって、培養液からのジアミンの分離が容易となると考えられる。 (a) Culturing step The method for producing diamine includes a culturing step of culturing the recombinant microorganism according to the above-described embodiment. By culturing in this step, a culture solution containing the cells is obtained. In the culturing step, the recombinant microorganism may be cultured in a culture solution containing an inorganic salt. Inorganic salts are hydrochlorides, sulfates, phosphates, carbonates, hydrofluorides, etc. of metal elements, such as sodium chloride, lithium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, and sodium carbonate. Among them, sodium sulfate and sodium carbonate are preferred. Without intending to be bound by any theory, when an inorganic salt is added to an aqueous solution, its strong hydration force fixes water molecules as water of hydration, thus reducing the amount of water molecules required for hydration of diamines. It is thought that phase separation occurs. The Hoffmeister series is an index indicating the strength of salting-out of inorganic salts, and salts composed of a combination of anions and cations (especially metal ions) that strongly cause salting-out, which are shown in the Hoffmeister series, are preferred. Diamines are produced by culturing in a culture solution containing inorganic salts, but in the presence of a high concentration of inorganic salts, phase separation of the diamine aqueous solution occurs from the culture solution. Therefore, it is considered that the inclusion of an inorganic salt in the culture solution facilitates the separation of the diamine from the culture solution.
　この場合、組換え組成物は、例えば５質量％以上の無機塩を含有する培養液で培養され、好ましくは１０質量％以上の無機塩を含有する培養液で培養される。別の態様において、組換え組成物は、例えば５～２０質量％、好ましくは１０～２０質量％の無機塩を含有する培養液で培養される。 In this case, the recombinant composition is cultured in a culture medium containing, for example, 5% by mass or more of inorganic salt, preferably 10% by mass or more of inorganic salt. In another embodiment, the recombinant composition is cultured in a medium containing, for example, 5-20%, preferably 10-20% by weight of inorganic salts.
　無機塩を含んだ相は、以下で説明する分離工程によって培養液から分離された後、培養液として再利用することができる。塩を高濃度で含んだ排水は処理にコストがかかるが、再利用によってコストを抑えることができる。また、精製プロセスにおける負荷、特に、相分離による脱水濃縮の負荷を低減することも可能である。例えば、ジアミンの分離のために強塩基を添加する先行技術も提案されているが、強塩基を添加する場合、無機塩添加で生ずる相分離は起こらず、ジアミン精製にあたり、培地からの脱水濃縮倍率が高くなり、さらに、溶媒抽出工程も追加的に必要となる。上記のように、無機塩を培養液に添加すれば、精製プロセスにおいて、脱水濃縮の負荷を低減することができ、かつ、溶媒抽出のための追加工程も不要となる。 The phase containing inorganic salts can be reused as a culture solution after being separated from the culture solution by the separation process described below. Wastewater with a high concentration of salt is costly to treat, but recycling can reduce costs. It is also possible to reduce the load in the purification process, especially the load of dehydration and concentration due to phase separation. For example, a prior art has been proposed in which a strong base is added for diamine separation. is higher, and an additional solvent extraction step is required. As described above, by adding an inorganic salt to the culture solution, it is possible to reduce the load of dehydration and concentration in the purification process, and an additional step for solvent extraction becomes unnecessary.
　（ｂ）反応工程
　本工程は、ジアミンの前駆体を組換え微生物に接触させる工程であり、ジアミン前駆体から目的のジアミンを生成する。ジアミン前駆体との接触は、例えば、前記培養工程においてまたは当該工程の後に行ってもよい。 (b) Reaction step This step is a step of contacting a diamine precursor with a recombinant microorganism to produce the desired diamine from the diamine precursor. Contact with the diamine precursor may, for example, take place during or after the culturing step.
　例えば、ジアミンが１，５－ペンタメチレンジアミンである場合、組換え微生物をリジンと接触させることにより、組換え微生物によって生産されたリジンデカルボキシラーゼにより、リジンが脱炭酸され、１，５－ペンタメチレンジアミンが生成する。 For example, when the diamine is 1,5-pentamethylenediamine, contacting the recombinant microorganism with lysine causes the lysine to be decarboxylated by lysine decarboxylase produced by the recombinant microorganism to produce 1,5-pentamethylenediamine. A diamine is formed.
　一態様において、本工程では、前記培養工程で得られた前記培養液および／または前記菌体を、５質量％以上の無機塩とリジンとを含有する水溶液と接触させて１，５－ペンタンジアミンを含む反応液を得る。例えば、本工程では、培養工程で得られた菌体を含む培養液を、および／または、培養工程で得られた培養液から遠心分離等によって上清を除去した菌体を、無機塩とリジンとを含有する水溶液と接触させ反応液を得る。 In one aspect, in this step, the culture solution and/or the bacterial cells obtained in the culture step are brought into contact with an aqueous solution containing 5% by mass or more of an inorganic salt and lysine to obtain 1,5-pentanediamine. Obtain a reaction solution containing For example, in this step, the culture solution containing the cells obtained in the culture step, and/or the cells obtained by removing the supernatant from the culture solution obtained in the culture step by centrifugation or the like, is mixed with an inorganic salt and lysine. is brought into contact with an aqueous solution containing and to obtain a reaction liquid.
　別の態様において、上記培養工程と反応工程とは、同一工程内で行われてもよい。例えば、ジアミンが１，５－ペンタメチレンジアミンである場合、組換え微生物を培養する培養液中に、無機塩とリジンとを含有する水溶液を添加してもよい。また、例えば、発酵によってリジンを生産する菌と、上記本発明にかかる組換え微生物とを共培養してもよい。これらを共培養することによって、前記菌によって生産されたリジンを、本発明にかかる組換え組成物の生産したリジンデカルボキシラーゼによって、効率的に１，５－ペンタメチレンジアミンに変換することができる。 In another aspect, the culture step and the reaction step may be performed in the same step. For example, when the diamine is 1,5-pentamethylenediamine, an aqueous solution containing an inorganic salt and lysine may be added to the culture medium for culturing the recombinant microorganism. Alternatively, for example, a bacterium that produces lysine by fermentation and the recombinant microorganism according to the present invention may be co-cultivated. By cocultivating them, the lysine produced by the fungus can be efficiently converted to 1,5-pentamethylenediamine by the lysine decarboxylase produced by the recombinant composition of the present invention.
　（無機塩の添加）
　無機塩は、あらかじめ培養液中に存在していてもよい。即ち、上記培養工程に関連して説明したとおり、本発明にかかる組換え微生物を、無機塩を含有する培養液で培養していてもよい。この場合、組換え組成物は、例えば５質量％以上の無機塩を含有する培養液で培養され、好ましくは１０質量％以上の無機塩を含有する培養液で培養される。別の態様では、組換え組成物は、例えば５～２０質量％、好ましくは１０～２０質量％の無機塩を含有する培養液で培養される。 (Addition of inorganic salt)
The inorganic salt may be present in advance in the culture medium. That is, as explained in relation to the culture step, the recombinant microorganism according to the present invention may be cultured in a culture solution containing inorganic salts. In this case, the recombinant composition is cultured in a culture medium containing, for example, 5% by mass or more of inorganic salt, preferably 10% by mass or more of inorganic salt. In another aspect, the recombinant composition is cultured in a medium containing, for example, 5-20% by weight, preferably 10-20% by weight of inorganic salts.
　無機塩が培養液に添加される場合、培養液中の無機塩の濃度が、１００～２００ｇ／Ｌ、好ましくは１５０～２００ｇ／Ｌ、より好ましくは１６０～２００ｇ／Ｌ、さらにより好ましくは２００ｇ／Ｌとなるように添加される。 When an inorganic salt is added to the culture medium, the concentration of the inorganic salt in the culture medium is 100 to 200 g/L, preferably 150 to 200 g/L, more preferably 160 to 200 g/L, and even more preferably 200 g/L. L is added.
　あるいは、ジアミンが１，５－ペンタメチレンジアミンである場合、無機塩は、反応工程において、リジンと共に、水溶液として、培養工程で得られた培養液および／または菌体と接触させてもよい。一態様において、無機塩は、培養工程および反応工程の両方の工程で添加されてもよい。別の態様では、無機塩は、培養工程および／または反応工程の他、以下で説明するような、ジアミンの製造方法における工程の１または複数で添加されてもよい。 Alternatively, when the diamine is 1,5-pentamethylenediamine, the inorganic salt may be brought into contact with the culture solution and/or the cells obtained in the culture step as an aqueous solution together with lysine in the reaction step. In one aspect, inorganic salts may be added during both the culturing step and the reaction step. In another aspect, inorganic salts may be added in one or more of the steps in the process for producing diamines, such as those described below, in addition to the culturing and/or reaction steps.
　無機塩は、炭酸ナトリウムまたは硫酸ナトリウムであり、より好ましくは硫酸ナトリウムである。 The inorganic salt is sodium carbonate or sodium sulfate, more preferably sodium sulfate.
　無機塩を添加することにより、以下で説明する分離工程において、ジアミンを含む相と、無機塩を含む水相とを相分離させることができ、ジアミンの分離が容易となる。また、本発明にかかる組換え微生物が、好塩性の性質を有する微生物である場合、このように高濃度の無機塩を添加した場合でも微生物の生育が阻害されることがないので、ジアミンの産生を妨げることなくジアミンを含む相の分離を促進することができ、ジアミンを容易に単離することができるという利点がある。 By adding an inorganic salt, a phase containing a diamine and an aqueous phase containing an inorganic salt can be phase-separated in the separation step described below, facilitating separation of the diamine. In addition, when the recombinant microorganism according to the present invention is a microorganism having halophilic properties, the growth of the microorganism is not inhibited even when such a high concentration of inorganic salt is added. The advantage is that the separation of the diamine-containing phase can be facilitated without interfering with the production, and the diamine can be easily isolated.
　（ｃ）除去工程
　上記製造方法は、前記培養液または反応液から前記組換え微生物を除去する除去工程を更に含んでもよい。当該工程は例えば、前記培養工程または反応工程によりジアミンを生産した後に、例えば遠心分離および／または濾過処理によって行われる。当該工程によって、培養液または反応液に含まれる菌体などの固形分を除去できる。また、ろ過処理時に限外ろ過膜を使用することで、多糖類、タンパク質などを含む任意の分子量以上の高分子化合物を除去することができる。 (c) Removal Step The production method may further include a removal step of removing the recombinant microorganism from the culture solution or reaction solution. The step is performed, for example, by centrifugation and/or filtration after diamine is produced by the culture step or reaction step. Through this step, solids such as bacterial cells contained in the culture solution or reaction solution can be removed. In addition, by using an ultrafiltration membrane at the time of filtration, it is possible to remove high-molecular weight compounds having an arbitrary molecular weight or higher, including polysaccharides and proteins.
　（ｄ）濃縮工程
　当該工程では、前記培養液または反応液が濃縮される。濃縮工程は例えば、前記除去工程により前記組換え微生物を除去した後に、例えばエバポレーターなどを用いて培養上清を濃縮することにより行われる。ジアミンを含む培養液を濃縮することで、ジアミンおよび無機塩の濃度が上昇し、分離効率を一層向上させることが期待できる。 (d) Concentration step In this step, the culture solution or reaction solution is concentrated. The concentration step is performed, for example, by concentrating the culture supernatant using, for example, an evaporator after removing the recombinant microorganism by the removal step. By concentrating the diamine-containing culture medium, the concentrations of the diamine and the inorganic salt are increased, which is expected to further improve the separation efficiency.
　ジアミンが前記培養工程及び／又は反応工程により生産した炭酸塩、重炭酸塩、ビス重炭酸塩、カルバミン酸塩、及びビスカルバミン酸塩からなる群より選択される１種以上の形態である場合、前記濃縮工程において、当該塩形態のジアミンを遊離塩基及び二酸化炭素に転換させ、二酸化炭素を分離させる。例えば、ジアミンが１，５－ペンタメチレンジアミンである場合、前記培養工程により生産した炭酸塩、重炭酸塩、ビス重炭酸塩、カルバミン酸塩、及びビスカルバミン酸塩からなる群より選択される１種以上の形態である１，５－ペンタメチレンジアミンを、前記濃縮工程において、遊離塩基及び二酸化炭素に転換させ、二酸化炭素を分離させる。 When the diamine is in one or more forms selected from the group consisting of carbonate, bicarbonate, bisbicarbonate, carbamate, and biscarbamate produced by the culturing step and/or reaction step, In the concentration step, the salt form of the diamine is converted to the free base and carbon dioxide, which is separated. For example, when the diamine is 1,5-pentamethylenediamine, 1 selected from the group consisting of carbonate, bicarbonate, bisbicarbonate, carbamate, and biscarbamate produced by the culture step 1,5-Pentamethylenediamine, in more than one form, is converted to the free base and carbon dioxide in the concentration step, and the carbon dioxide is separated.
　（ｅ）ｐＨ調整工程
　ｐＨ調整工程では、前記培養液または前記反応液のｐＨを１２以上に調整する。例えば、本工程では、前記濃縮工程により培養液または反応液を濃縮した後に、濃縮した培養液または反応液のｐＨを１２以上に調整する。あるいは濃縮工程における二酸化炭素の分離によるｐＨ上昇によって、ｐＨが１２以上になっていることを確認する。 (e) pH adjustment step In the pH adjustment step, the pH of the culture solution or the reaction solution is adjusted to 12 or more. For example, in this step, after concentrating the culture solution or reaction solution in the concentration step, the pH of the concentrated culture solution or reaction solution is adjusted to 12 or higher. Alternatively, it is confirmed that the pH is 12 or higher due to the pH increase due to the separation of carbon dioxide in the concentration step.
　（ｆ）分離工程
　本工程では、培養液または反応液から、ジアミンを含む相を分離する。分離工程では、アルカリ化合物を添加することを含まない。ここでいうアルカリ化合物は、その水溶液が塩基性を示し、それを添加することによってｐＨ値を上昇させる作用を有する化合物を指し、特に無機のアルカリ化合物である。金属元素の水酸化物、および、水素イオンを受容可能な無機物質が該当するが、無機のアルカリ化合物としては、例えば、水酸化カリウム、水酸化ナトリウム、水酸化カルシウム、水酸化リチウム、アンモニア、水酸化マグネシウム、水酸化アルミニウム、水酸化マンガン、水酸化鉄、水酸化コバルト、水酸化銅、水酸化亜鉛、水酸化バリウムなどが挙げられる。アルカリ化合物を添加しないことにより、培地成分以外にアルカリ化合物を使用しないことによるコスト低減が達成される。また、高ｐＨに起因するジアミン類を分離した後の、無機塩を含む相のｐＨが下がることで、培地としてのリサイクル使用も可能となる。培養液または反応液に無機塩が含まれている場合、本分離工程では、ジアミンを含む相と、無機塩を含む水相とを相分離する。 (f) Separation Step In this step, a phase containing diamine is separated from the culture solution or reaction solution. The separation step does not involve adding alkaline compounds. The alkali compound as used herein refers to a compound whose aqueous solution exhibits basicity and has the effect of increasing the pH value by adding it, and is particularly an inorganic alkali compound. Hydroxides of metal elements and inorganic substances capable of accepting hydrogen ions fall under this category. Examples of inorganic alkaline compounds include potassium hydroxide, sodium hydroxide, calcium hydroxide, lithium hydroxide, ammonia, and water. magnesium oxide, aluminum hydroxide, manganese hydroxide, iron hydroxide, cobalt hydroxide, copper hydroxide, zinc hydroxide, barium hydroxide and the like. By not adding alkaline compounds, cost reduction is achieved by not using alkaline compounds other than medium components. In addition, the pH of the inorganic salt-containing phase after separation of diamines due to high pH is lowered, so that it can be recycled as a medium. When the culture solution or reaction solution contains an inorganic salt, in this separation step, a phase containing a diamine and an aqueous phase containing an inorganic salt are phase-separated.
　相分離は、前記培養液もしくは反応液に有機溶媒を添加することにより、または、培養液もしくは反応液を有機溶媒と接触させることにより行ってもよい。有機溶媒は、例えば、ｎ－ヘキサン、ｎ－ブタノール、及び２－エチル－１－ヘキサノールからなる群より選択される１種以上である。例えば、ジアミンが１，５－ペンタメチレンジアミンの場合には、ｎ－ブタノールを用いることが好ましく、ジアミンがＨＭＤＡの場合、２－エチル－１－ヘキサノールを用いることが好ましい。 Phase separation may be performed by adding an organic solvent to the culture solution or reaction solution, or by bringing the culture solution or reaction solution into contact with an organic solvent. The organic solvent is, for example, one or more selected from the group consisting of n-hexane, n-butanol, and 2-ethyl-1-hexanol. For example, when the diamine is 1,5-pentamethylenediamine, it is preferred to use n-butanol, and when the diamine is HMDA, it is preferred to use 2-ethyl-1-hexanol.
　上記のとおり、高塩濃度の無機塩（例えば炭酸ナトリウムおよび硫酸ナトリウムなど）によってジアミン類の相分離が促進されるが、さらに有機溶媒が添加されて相分離が促進されてもよい。有機溶媒を添加することによって、ジアミンの水相から有機相への抽出を促進して有機相への移行率を高め、収率をさらに向上させることができる。 As described above, the phase separation of diamines is promoted by inorganic salts of high salt concentration (for example, sodium carbonate and sodium sulfate), but an organic solvent may be added to further promote phase separation. By adding an organic solvent, the extraction of the diamine from the aqueous phase to the organic phase can be promoted to increase the rate of transfer to the organic phase, thereby further improving the yield.
　（ｇ）水相回収工程および再利用工程
　本工程では、無機塩を含む相（例えば、無機塩および水を含む）を回収し、および／または、回収した相を培養液として再利用する。本発明にかかる組換え微生物は好塩性を有するため、無機塩を含んだ培養液中でも生育が阻害されることなく、ジアミンを産生することができる。また、無機塩が含まれることで、相分離が促進されるので、ジアミンを分離することが容易になる。さらには、無機塩を高濃度で含有する水の処理にはコストがかかる可能性があるが、上記のように微生物の培地として再利用に供することにより、排水処理コストを低減することができる。 (g) Aqueous Phase Recovery Step and Reuse Step In this step, a phase containing an inorganic salt (for example, containing an inorganic salt and water) is recovered and/or the recovered phase is reused as a culture solution. Since the recombinant microorganism according to the present invention has halophilic properties, it can produce diamine without inhibiting its growth even in a culture solution containing inorganic salts. In addition, the inclusion of the inorganic salt promotes phase separation, making it easier to separate the diamine. Furthermore, treatment of water containing a high concentration of inorganic salts may be costly, but by reusing the water as a culture medium for microorganisms as described above, wastewater treatment costs can be reduced.
　（ｈ）精製工程
　本工程では、培養物から得られたジアミンを精製する。培養物からジアミン、例えば１，５－ペンタンジアミンを精製する方法は当業者に公知である。原核微生物宿主細胞の形質転換体またはゲノム編集体の場合、１，５－ペンタンジアミンは培養上清中または菌体内に存在するが、必要であれば培養菌体から抽出してもよい。培養菌体から抽出する場合、例えば、培養物を遠心分離して上清と菌体を分離し、ホモジナイザーを利用しつつ、界面活性剤、有機溶媒、酵素等により菌体を破壊し得る。培養上清、及び場合により菌体抽出液から精製する方法としては、ｐＨ調整等によるタンパク質沈澱を利用した除タンパク処理、活性炭を利用した不純物の吸着による除去、イオン交換樹脂等を利用したイオン性物質の吸着による除去を実施した後に、公知の溶媒を使用した抽出、蒸留等で精製される。勿論、製品により目的とされる純度に応じて一部の工程を削除したり、或いはクロマトグラフィーなどの追加の精製工程を実施したりしてもよいことは言うまでもない。 (h) Purification step In this step, the diamine obtained from the culture is purified. Methods for purifying diamines, such as 1,5-pentanediamine, from cultures are known to those skilled in the art. In the case of transformants or genome editors of prokaryotic microbial host cells, 1,5-pentanediamine is present in the culture supernatant or within the cells, but may be extracted from the cultured cells if necessary. When extracting from cultured cells, for example, the culture may be centrifuged to separate the supernatant from the cells, and the cells may be disrupted with a surfactant, organic solvent, enzyme or the like using a homogenizer. Methods for purifying the culture supernatant and, in some cases, the bacterial cell extract include deproteinization using protein precipitation by pH adjustment, etc., removal of impurities by adsorption using activated carbon, and ionic purification using ion exchange resins. After removing the substance by adsorption, it is purified by extraction using a known solvent, distillation, or the like. Of course, it goes without saying that some steps may be omitted or additional purification steps such as chromatography may be performed depending on the purity desired for the product.
　本発明の更に別の実施形態は、先述の組換え組成物を用いて得られた培養物からジアミンを精製する方法にも関する。当該精製方法は、ジアミンの製造方法について記載した上記の各工程を、単独でまたは組み合わせて含んでよい。 Yet another embodiment of the present invention also relates to a method of purifying diamines from cultures obtained using the aforementioned recombinant compositions. The purification method may include, singly or in combination, each of the steps described above for the diamine production method.
　一般的に、従来の方法で用いられていた大腸菌およびコリネ型細菌は、ｐＨ９以上の環境では生育が著しく阻害されていたため、例えば、リジンを１，５－ペンタメチレンジアミンへ変換することで培地中のｐＨが上昇すると、酸溶液を適宜加えることでｐＨを微生物の生育を阻害しない範囲に制御する必要があった。しかしながら、本発明にかかる組換え微生物は、アルカリ環境下でも生育できる好アルカリ性を有するため、当該微生物をジアミン生産に用いることで、従来技術のように、酸溶液添加によるｐＨ調整を行う必要がなく、ジアミンの製造プロセスの煩雑化を回避することができる。また、本発明にかかる組換え微生物が好塩性である場合、塩が含まれる培養液中でも生育可能である。そのため、分離工程で添加した塩を含む廃液を当該微生物の培養に再利用することができるため、排水処理コストを低減することができる。また、ジアミンの分離のために強塩基を添加する先行技術と比較して、無機塩を培養液に添加することで、精製プロセスにおいて、脱水濃縮の負荷を低減することができ、かつ、溶媒抽出のための追加工程も不要となる。 In general, the growth of Escherichia coli and coryneform bacteria used in conventional methods is significantly inhibited in an environment of pH 9 or higher. When the pH of the solution rises, it is necessary to control the pH within a range that does not inhibit the growth of microorganisms by appropriately adding an acid solution. However, since the recombinant microorganism according to the present invention has alkalophilicity that allows it to grow even in an alkaline environment, by using the microorganism for diamine production, there is no need to adjust the pH by adding an acid solution as in the prior art. , the complication of the diamine production process can be avoided. In addition, if the recombinant microorganism according to the present invention is halophilic, it can grow even in a salt-containing culture medium. Therefore, the waste liquid containing the salt added in the separation step can be reused for culturing the microorganism, and the waste water treatment cost can be reduced. In addition, compared to the prior art in which a strong base is added for diamine separation, adding an inorganic salt to the culture solution can reduce the load of dehydration concentration in the purification process, and solvent extraction An additional process for is also unnecessary.
＜２＞発明Ｂ：Ｎ－アセチルジアミン生産を抑制した好塩性および／または好アルカリ性の組換え微生物 <2> Invention B: Halophilic and/or alkalophilic recombinant microorganism that suppresses N-acetyldiamine production
　本発明の遺伝子組換え微生物は、ジアミン生産能を有する、好塩性および／または好アルカリ性の宿主微生物に対して、Ｎ－アセチル化酵素を抑制する１以上の遺伝子改変を行ったものである。 The genetically modified microorganism of the present invention is a halophilic and/or alkalophilic host microorganism with diamine-producing ability that has undergone one or more genetic alterations to suppress N-acetylase.
　自然界には、ｐＨ９以上のアルカリ環境でも生育することができる好アルカリ性の性質、０．２Ｍ以上のＮａＣｌ濃度の環境でも生育することができる好塩性の性質、またはこれら両者の性質を併せ持つ微生物が存在する。本発明に関連して、宿主微生物として利用できる好アルカリ性微生物とは、多様な分布を示す極限環境微生物の一種であり、ｐＨが９以上の環境下でも生育可能な微生物の総称である。これらは、ｐＨ９以上の環境でのみ生育できる絶対好アルカリ性微生物と、ｐＨ９以上に至適生育ｐＨを示すものの、ｐＨ９未満でも生育することができる通性好アルカリ性微生物とに分類される。その中のいくつかはｐＨ１２以上の強アルカリ性環境でも生育することができる。これらのいずれもが、好アルカリ性微生物であり、本発明において、宿主微生物となり得る。 In the natural world, there are microorganisms that have alkalophilic properties that allow them to grow in an alkaline environment with a pH of 9 or higher, halophilic properties that allow them to grow in an environment with a NaCl concentration of 0.2M or higher, or those that have both of these properties. exist. In relation to the present invention, alkalophilic microorganisms that can be used as host microorganisms are a kind of extremophilic microorganisms that exhibit diverse distributions and are a general term for microorganisms that can grow even in an environment with a pH of 9 or higher. These are classified into obligate alkalophilic microorganisms, which can grow only in an environment of pH 9 or higher, and facultative alkalophilic microorganisms, which have an optimum growth pH of 9 or higher but can grow even below pH 9. Some of them can grow even in a strong alkaline environment with a pH of 12 or higher. All of these are alkalophilic microorganisms and can be host microorganisms in the present invention.
　本発明に関連して、宿主微生物として利用できる好塩性微生物とは、高濃度の塩ストレスに対応できる微生物の総称である。これらは、最適増殖塩濃度による菌の分類では、塩化ナトリウム０～０．２Ｍが最適増殖塩濃度である非好塩菌、０．２～０．５Ｍが最適増殖塩濃度である低度好塩菌、０．５～２．５Ｍが最適増殖塩濃度である中度好塩菌、２．５～５．２Ｍが最適増殖塩濃度である高度好塩菌に分類される。これらのいずれもが、好塩性微生物であり、本発明において、宿主微生物となり得る。 In relation to the present invention, halophilic microorganisms that can be used as host microorganisms is a general term for microorganisms that can cope with high-concentration salt stress. These are non-halophilic bacteria whose optimum growth salt concentration is 0-0.2M sodium chloride, and low halophilic bacteria whose optimum growth salt concentration is 0.2-0.5M. Moderate halophiles whose optimum growth salt concentration is 0.5 to 2.5M, and high halophiles whose optimum growth salt concentration is 2.5 to 5.2M. All of these are halophilic microorganisms and can be host microorganisms in the present invention.
　本発明の宿主微生物として利用できる、好塩性、好アルカリ性、または両方の性質を持つ微生物には、様々な微生物が含まれるが、その非限定的な例としては、バチルス属、ハロモナス属、ハロバクテロイデス属、サリニバクター属、アルカリファイラス属、クロストリジウム属、アンエアロブランカ属の細菌等が挙げられる。本発明の宿主微生物は、好ましくは、バチルス属の細菌である。バチルス属の微生物の中でも、バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｅｕｄｏｆｉｒｍｕｓ）、バチルス・ハロデュランス(Ｂａｃｉｌｌｕｓ　ｈａｌｏｄｕｒａｎｓ)、バチルス・マーマエンシス(Ｂａｃｉｌｌｕｓ　ｍａｒｍａｒｅｎｓｉｓ)が好ましく、バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｕｅｄｏｆｉｒｍｕｓ）がより好ましい。 Halophilic, alkalophilic, or both microorganisms that can be used as host microorganisms of the present invention include various microorganisms, non-limiting examples of which include Bacillus, Halomonas, Halo Bacteroides, Salinibacter, Alkalinephilus, Clostridium, Anaeroblanca, and the like. The host microorganism of the invention is preferably a bacterium of the genus Bacillus. Among microorganisms belonging to the genus Bacillus, Bacillus pseudofirmus, Bacillus halodurans, and Bacillus marmarensis are preferred, and Bacillus pseudofirmus is more preferred.
　化合物の生合成に一般的に利用される大腸菌およびコリネ型細菌は、ｐＨ９以上の環境では生育が著しく阻害されているため、例えば、リジンを１，５－ペンタメチレンジアミンへ変換することで培地中のｐＨが上昇すると、酸溶液を適宜加えることでｐＨを微生物の生育を阻害しない範囲に制御する必要があった。しかしながら、本発明にかかる組換え微生物は、アルカリ環境下でも生育できる好アルカリ性を有するため、当該微生物をジアミン生産に用いることで、従来のように、酸溶液添加によるｐＨ調整を行う必要がなく、ジアミンの製造プロセスの煩雑化を回避することができる。また、本発明にかかる組換え微生物が好塩性である場合、塩が含まれる培養液中でも生育可能である。そのため、培養液または混合液（反応液）からジアミンを含む相を分離する分離工程で添加した塩を含む廃液を、本発明にかかる微生物の培養に再利用することができるため、排水処理コストを低減することができる。 The growth of Escherichia coli and coryneform bacteria, which are commonly used for the biosynthesis of compounds, is significantly inhibited in an environment of pH 9 or higher. When the pH of the solution rises, it is necessary to control the pH within a range that does not inhibit the growth of microorganisms by appropriately adding an acid solution. However, since the recombinant microorganism according to the present invention has alkalophilicity that allows it to grow even in an alkaline environment, by using the microorganism for diamine production, there is no need to adjust the pH by adding an acid solution as in the conventional method, It is possible to avoid complication of the diamine manufacturing process. In addition, if the recombinant microorganism according to the present invention is halophilic, it can grow even in a salt-containing culture medium. Therefore, the waste liquid containing the salt added in the separation step of separating the diamine-containing phase from the culture solution or the mixed solution (reaction solution) can be reused for culturing the microorganisms according to the present invention, thus reducing the wastewater treatment cost. can be reduced.
　ジアミン生産能を有する好塩性および／または好アルカリ性の微生物を宿主微生物として使用し、Ｎ－アセチル化酵素を抑制する１以上の遺伝子改変を行うことにより、得られた組換え微生物において、副生物であるＮ－アセチルジアミン生産の抑制が可能となる。 A by-product in a recombinant microorganism obtained by using a halophilic and/or alkalophilic microorganism having diamine-producing ability as a host microorganism and performing one or more genetic modifications to suppress N-acetylase It is possible to suppress the production of N-acetyldiamine.
　すなわち、ジアミン生産能を有する好塩性および／または好アルカリ性の微生物において、Ｎ－アセチルジアミン生産の抑制することにより、酵素変換工程または培養工程においてＮ－アセチルジアミン生産を抑制することができる。好適な態様においては、副生物の産生を抑制することによって、目的化合物であるジアミン化合物を効率よく生産することができる。 That is, by suppressing N-acetyldiamine production in halophilic and/or alkalophilic microorganisms having diamine-producing ability, N-acetyldiamine production can be suppressed in the enzymatic conversion process or culture process. In a preferred embodiment, the target diamine compound can be efficiently produced by suppressing the production of by-products.
　本明細書において、「Ｎ－アセチル化酵素」とは、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素である。「Ｎ－アセチル化酵素」の語は、「Ｎ－アセチル転移酵素」および「Ｎ－アセチルトランスフェラーゼ」の語と同義であり、本明細書において、これらの語は互換可能に使用される。Ｎ－アセチル化酵素は、アミノ酸のＮ末端をアセチル化する反応を触媒する酵素であり、ジアミン生産経路においては、副反応として、ジアミンをＮ－アセチル化してＮ－アセチルジアミンを生成する反応を触媒する。 As used herein, the term "N-acetylating enzyme" refers to an enzyme that N-acetylates a diamine compound to produce an N-acetyldiamine compound. The term "N-acetyltransferase" is synonymous with the terms "N-acetyltransferase" and "N-acetyltransferase" and these terms are used interchangeably herein. N-acetyltransferase is an enzyme that catalyzes a reaction that acetylates the N-terminus of an amino acid. In the diamine production pathway, as a side reaction, it catalyzes a reaction that N-acetylates diamine to produce N-acetyldiamine. do.
　本発明では、上記のとおり、好塩性および／または好アルカリ性の宿主微生物において、Ｎ－アセチル化酵素を抑制する１以上の遺伝子改変が行われており、したがって、本発明にかかる組換え微生物は、Ｎ－アセチル化酵素を抑制する１以上の遺伝子改変を含むものである。当該遺伝子改変は、
・前記Ｎ－アセチル化酵素をコードする内因性遺伝子の発現を抑制する改変であるか、または、
・当該Ｎ－アセチル化酵素の活性が低下する改変である。 In the present invention, as described above, a halophilic and/or alkalophilic host microorganism is subjected to one or more genetic modifications that inhibit N-acetyltransferase. , containing one or more genetic modifications that inhibit N-acetyltransferase. The genetic modification is
- modification that suppresses the expression of the endogenous gene encoding the N-acetyltransferase, or
- A modification that reduces the activity of the N-acetyltransferase.
　微生物におけるＮ－アセチル化酵素の抑制は、例えば、イオンクロマトグラフィー等の分析方法を用いて、
・当該微生物の培養上清にＮ－アセチル体が検出されないこと、
・細胞破砕液を用いた酵素活性測定時に基質であるカダベリンが減少しないこと、および
・生成物であるＮ－アセチル体が検出されないこと
の少なくとも１つによって確認することができる。 Inhibition of N-acetyltransferase in microorganisms can be achieved by, for example, using analytical methods such as ion chromatography,
- no N-acetyl form is detected in the culture supernatant of the microorganism;
It can be confirmed by at least one of: - no decrease in cadaverine as a substrate and - no detection of the N-acetyl form as a product during measurement of enzymatic activity using a cell lysate.
　好ましい態様において、本発明にかかる組換え微生物は、Ｎ－アセチルジアミン化合物の生産能が、遺伝子改変を含まない非変異株の当該生産能と比較して、抑制されているか又は消失している。 In a preferred embodiment, the recombinant microorganism of the present invention has suppressed or eliminated N-acetyldiamine compound-producing ability compared to the non-mutant strain containing no genetic modification.
　Ｎ－アセチル化酵素を抑制する１以上の遺伝子改変は、Ｎ－アセチルジアミン生合成経路を抑制するための改変である。当該遺伝子改変は、例えば：
・宿主微生物において、Ｎ－アセチル化酵素をコードする、内因性遺伝子の一部もしくは全部を上記宿主微生物のゲノム配列から削除すること、
・宿主微生物において、Ｎ－アセチル化酵素の遺伝子配列に酵素機能が欠失するような変異を導入すること、
・Ｎ－アセチル化酵素のプロモーター部位および／またはＲＢＳ部位に置換、挿入、欠失などの変異を導入すること
のうちの１以上により行われる。 One or more genetic modifications that inhibit N-acetyltransferase are modifications to inhibit the N-acetyldiamine biosynthetic pathway. Such genetic modifications are for example:
- deleting part or all of the endogenous gene encoding the N-acetyltransferase in the host microorganism from the genome sequence of the host microorganism;
-Introducing a mutation in the gene sequence of the N-acetyltransferase in the host microorganism that results in a loss of enzymatic function;
- It is carried out by one or more of the methods of introducing mutations such as substitutions, insertions and deletions into the promoter site and/or the RBS site of the N-acetyltransferase.
　Ｎ－アセチルジアミン生合成経路を抑制するための改変は、例えば：
・宿主微生物において、Ｎ－アセチル化酵素をコードする、内因性遺伝子の一部もしくは全部を上記宿主微生物のゲノム配列から削除すること、
・宿主微生物において、Ｎ－アセチル化酵素の遺伝子配列に酵素機能が欠失するような変異を導入すること、
・Ｎ－アセチル化酵素のプロモーター部位および／またはＲＢＳ部位に置換、挿入、欠失などの変異を導入すること
のうちの１以上により行われる。 Modifications to inhibit the N-acetyldiamine biosynthetic pathway include, for example:
- deleting part or all of the endogenous gene encoding the N-acetyltransferase in the host microorganism from the genome sequence of the host microorganism;
-Introducing a mutation in the gene sequence of the N-acetyltransferase in the host microorganism that results in a loss of enzymatic function;
- It is carried out by one or more of the methods of introducing mutations such as substitutions, insertions and deletions into the promoter site and/or the RBS site of the N-acetyltransferase.
　上記のような操作により得られた組換え微生物においては、Ｎ－アセチル化酵素の生産が抑制される。すなわち、本発明にかかる組換え微生物は、Ｎ－アセチル化酵素の生産を抑制するように、１つ以上の遺伝子操作により改変されている。かかる改変により、本発明にかかる組換え微生物は、好ましくは、Ｎ－アセチルジアミン化合物の生産能が、前記遺伝子改変を含まない非変異株の当該生産能と比較して、抑制されているか又は消失している。 The production of N-acetyltransferase is suppressed in the recombinant microorganisms obtained by the above operations. That is, the recombinant microorganism according to the present invention has been modified by one or more genetic manipulations so as to suppress the production of N-acetyltransferase. Due to such modification, the recombinant microorganism of the present invention preferably has suppressed or eliminated N-acetyldiamine compound-producing ability compared to the non-mutant strain containing no genetic modification. is doing.
　前記遺伝子操作は、具体的には、例えば、下記（Ａ）、（Ｂ）、（Ｃ）及び（Ｄ）から成る群より選択される１以上の遺伝子操作であってよい。
（Ａ）前記宿主微生物に、前記Ｎ－アセチルトランスフェラーゼをコードする内因性遺伝子を欠失させる操作、
（Ｂ）前記宿主微生物内の前記Ｎ－アセチルトランスフェラーゼの内因性遺伝子のコピー数を低下させる操作、
（Ｃ）前記宿主微生物内の前記Ｎ－アセチルトランスフェラーゼの内因性遺伝子の発現調整領域に変異を導入する操作、および
（Ｄ）前記宿主微生物内の前記Ｎ－アセチルトランスフェラーゼの内因性遺伝子の発現調整領域を、低発現可能な外来調整領域で置換する操作。 Specifically, the genetic manipulation may be, for example, one or more genetic manipulations selected from the group consisting of (A), (B), (C) and (D) below.
(A) an operation of deleting an endogenous gene encoding the N-acetyltransferase from the host microorganism;
(B) reducing the copy number of the endogenous gene for said N-acetyltransferase in said host microorganism;
(C) an operation of introducing a mutation into the expression control region of the endogenous gene of the N-acetyltransferase in the host microorganism; and (D) an expression control region of the endogenous gene of the N-acetyltransferase in the host microorganism. is replaced with a low-expressible exogenous regulatory region.
　遺伝子改変には、例えば、宿主微生物の細胞内にゲノム配列に任意の外来の遺伝子配列を導入する技術、および、ゲノム配列から不要な遺伝子配列を取り除く技術を利用することができる。 For genetic modification, for example, techniques for introducing any foreign gene sequence into the genome sequence into the cells of the host microorganism and techniques for removing unnecessary gene sequences from the genome sequence can be used.
　具体的には、以下のような技術が挙げられる。
　（１）温度感受性プラスミドベクターとネガティブセレクションを組み合わせた染色体
上の遺伝子配列を任意に改変する技術、および、
　（２）ＣＲＩＳＰＲ／ＣＡＳ９による染色体上の遺伝子配列を任意に改変する技術。 Specifically, the following techniques are mentioned.
(1) A technique for arbitrarily modifying a gene sequence on a chromosome by combining a temperature-sensitive plasmid vector and negative selection, and
(2) A technique for arbitrarily modifying gene sequences on chromosomes by CRISPR/CAS9.
　上記で説明したベクターが導入された宿主微生物を培養する過程で、温度変化と培地組成変化により、相同組換えにより染色体の遺伝子配列が改変された遺伝子組換え株を獲得することができる。遺伝子組換え株を獲得する時の条件としては、培養温度が３７～４３℃以上であること、培地に１ｍＭ以上の４－クロロフェニルアラニンが含まれていることが好ましい。 　In the process of culturing the host microorganism into which the vector described above has been introduced, it is possible to obtain a genetically modified strain in which the gene sequence of the chromosome has been modified by homologous recombination due to changes in temperature and medium composition. As conditions for obtaining a genetically modified strain, it is preferable that the culture temperature is 37 to 43° C. or higher and that the medium contains 1 mM or higher 4-chlorophenylalanine.
　上記で説明したベクターを宿主微生物に導入する際に適用できる手法として、例えば、接合伝達、共沈、プロトプラスト融合、エレクトロポレーション、レトロウイルストランスフェクションなどの慣用のクローニング法およびトランスフェクション法が使用される。それらの例は、Ｇｅｎｅ　Ｃｌｏｎｉｎｇ　ａｎｄ　ＤＮＡ　Ａｎａｌｙｓｉｓ、Ｔ．Ａ．Ｂｒｏｗｎ、２０１６、またはＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ （Ｆｏｕｒｔｈ Ｅｄｉｔｉｏｎ）：Ｓａｍｂｒｏｏｋら、２０１２に記載されている。 Techniques that can be applied to introduce the vectors described above into host microorganisms include conventional cloning and transfection methods such as conjugative transfer, co-precipitation, protoplast fusion, electroporation, retroviral transfection, and the like. be. Examples thereof can be found in Gene Cloning and DNA Analysis, T.W. A. Brown, 2016, or Molecular Cloning: A Laboratory Manual (Fourth Edition): Sambrook et al., 2012.
　代表的なＮ－アセチルトランスフェラーゼの遺伝子として、バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｕｅｄｏｆｉｒｍｕｓ）のｙｊｂＣが挙げられる。バチルス・シュードフィラマスｙｊｂＣ酵素のアミノ酸配列を配列番号２３に、バチルス・シュードフィラマスｙｊｂＣ遺伝子（ＢｐＯＦ４＿０１９２５（ＧｅｎＢａｎｋ：ＡＤＣ４８４５２））の塩基配列を配列番号２４に示す（図３および図４）。 A typical N-acetyltransferase gene is yjbC of Bacillus pseudofirmus. The amino acid sequence of the Bacillus pseudofilamus yjbC enzyme is shown in SEQ ID NO: 23, and the nucleotide sequence of the Bacillus pseudofilamus yjbC gene (BpOF4_01925 (GenBank: ADC48452)) is shown in SEQ ID NO: 24 (FIGS. 3 and 4).
　一態様において、Ｎ－アセチル化酵素は、
　（Ａ－１）配列番号２３に示されるアミノ酸配列からなるか、
　（Ａ－２）配列番号２３に示されるアミノ酸配列と８０％以上、８５％以上、８８％以上、９０％以上、９３％以上、９５％以上、９７％以上、９８％以上または９９％以上の配列同一性を有するアミノ酸配列からなり、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するか、もしくは
　（Ａ－３）配列番号２３に示されるアミノ酸配列に対して１～１０個、１～７個、１～５個、または１～３個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有する。 In one aspect, the N-acetyltransferase is
(A-1) consisting of the amino acid sequence shown in SEQ ID NO: 23,
(A-2) 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the amino acid sequence shown in SEQ ID NO: 23 or (A-3) 1 to the amino acid sequence shown in SEQ ID NO: 23. consisting of an amino acid sequence in which ~10, 1-7, 1-5, or 1-3 amino acids have been deleted, substituted, inserted and/or added; It has enzymatic activity that produces diamine compounds.
　好ましい態様において、Ｎ－アセチル化酵素は、
　（Ａ－１）配列番号２３に示されるアミノ酸配列からなるか、
　（Ａ－２）配列番号２３に示されるアミノ酸配列と９０％以上の配列同一性を有するアミノ酸配列からなり、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するか、もしくは
　（Ａ－３）配列番号２３に示されるアミノ酸配列に対して１～１０個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有する。 In a preferred embodiment, the N-acetyltransferase is
(A-1) consisting of the amino acid sequence shown in SEQ ID NO: 23,
(A-2) consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23, and having an enzymatic activity to N-acetylate a diamine compound to produce an N-acetyldiamine compound; or (A-3) consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and/or added to the amino acid sequence shown in SEQ ID NO: 23, and N-acetylating the diamine compound to obtain N - have enzymatic activity to produce acetyldiamine compounds;
　より好ましい態様において、Ｎ－アセチル化酵素は、（Ａ－１）配列番号２３に示されるアミノ酸配列からなる。 In a more preferred embodiment, the N-acetyltransferase consists of (A-1) the amino acid sequence shown in SEQ ID NO:23.
　別の一態様において、Ｎ－アセチル化酵素は、
　（Ｂ－１）配列番号２４に示される塩基配列からなるＤＮＡ、
　（Ｂ－２）配列番号２４に示される塩基配列に相補的な塩基配列を有するＤＮＡと緊縮条件下でハイブリダイズし、かつ、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するタンパク質をコードするＤＮＡ、
　（Ｂ－３）配列番号２４に示される塩基配列と８０％以上、８５％以上、８８％以上、９０％以上、９３％以上、９５％以上、９７％以上、９８％以上または９９％以上の配列同一性を有する塩基配列からなり、かつ、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するタンパク質をコードするＤＮＡ、
　（Ｂ－４）配列番号２４に示される塩基配列によりコードされるタンパク質のアミノ酸配列に対して１～１０個、１～７個、１～５個、または１～３個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなるタンパク質をコードするＤＮＡであって、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するタンパク質をコードするＤＮＡ、もしくは
　（Ｂ－５）配列番号２４に示される塩基配列の縮重異性体からなるＤＮＡ
にコードされる。 In another aspect, the N-acetyltransferase is
(B-1) DNA consisting of the base sequence shown in SEQ ID NO: 24,
(B-2) an enzyme that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 24 and N-acetylates a diamine compound to produce an N-acetyldiamine compound DNA encoding a protein having activity;
(B-3) 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more of the base sequence shown in SEQ ID NO: 24 DNA encoding a protein consisting of a nucleotide sequence having sequence identity and having an enzymatic activity to N-acetylate a diamine compound to produce an N-acetyldiamine compound;
(B-4) deletion of 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids from the amino acid sequence of the protein encoded by the nucleotide sequence shown in SEQ ID NO: 24; A DNA encoding a protein consisting of a substituted, inserted and/or added amino acid sequence, the DNA encoding a protein having the enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound, or B-5) DNA consisting of a degenerate isomer of the base sequence shown in SEQ ID NO: 24
coded to
　好ましい態様において、Ｎ－アセチル化酵素は、
　（Ｂ－１）配列番号２４に示される塩基配列からなるＤＮＡ、
　（Ｂ－２）配列番号２４に示される塩基配列に相補的な塩基配列を有するＤＮＡと緊縮条件下でハイブリダイズし、かつ、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するタンパク質をコードするＤＮＡ、
　（Ｂ－３）配列番号２４に示される塩基配列と９０％以上の配列同一性を有する塩基配列からなり、かつ、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するタンパク質をコードするＤＮＡ、
　（Ｂ－４）配列番号２４に示される塩基配列によりコードされるタンパク質のアミノ酸配列に対して１～１０個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなるタンパク質をコードするＤＮＡであって、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するタンパク質をコードするＤＮＡ、もしくは
　（Ｂ－５）配列番号２４に示される塩基配列の縮重異性体からなるＤＮＡ
にコードされる。 In a preferred embodiment, the N-acetyltransferase is
(B-1) DNA consisting of the base sequence shown in SEQ ID NO: 24,
(B-2) an enzyme that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 24 and N-acetylates a diamine compound to produce an N-acetyldiamine compound DNA encoding a protein having activity;
(B-3) Consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 24, and having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound DNA that encodes a protein,
(B-4) Encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and/or added to the amino acid sequence of the protein encoded by the nucleotide sequence shown in SEQ ID NO: 24 (B-5) degenerate isomerism of the nucleotide sequence shown in SEQ ID NO: 24 body DNA
coded to
　本明細書において、「緊縮条件」とは、例えば、「１ｘＳＳＣ、０．１％ＳＤＳ、６０℃」程度の条件であり、より厳しい条件としては「０．１ｘＳＳＣ、０．１％ＳＤＳ、６０℃」程度の条件であり、さらに厳しい条件としては「０．１ｘＳＳＣ、０．１％ＳＤＳ、６８℃」程度の条件である。 As used herein, “stringent conditions” are, for example, conditions of about “1×SSC, 0.1% SDS, 60° C.”, and more stringent conditions are “0.1×SSC, 0.1% SDS, 60° C. and a more severe condition is about "0.1 x SSC, 0.1% SDS, 68°C".
　より好ましい態様において、Ｎ－アセチル化酵素は、（Ｂ－１）配列番号２４に示される塩基配列からなるＤＮＡにコードされる。 In a more preferred embodiment, the N-acetyltransferase is encoded by (B-1) DNA consisting of the nucleotide sequence shown in SEQ ID NO:24.
　なお、本明細書において、参照アミノ酸配列に対する比較アミノ酸配列の「配列同一性」の割合（％）は、これら２つの配列間の同一性が最大となるように配列を整列させ、必要であれば２つの配列の一方または双方にギャップが導入されたときの、参照配列中のアミノ酸残基と同一である、比較配列中のアミノ酸残基の百分率として定義される。このとき、保存的置換は配列同一性の一部として考慮しない。配列同一性は、公に入手可能なコンピュータソフトウェアを使用することによって決定することができ、例えば、ＢＬＡＳＴ（登録商標。以下、省略。）（Ｂａｓｉｃ　Ｌｏｃａｌ　Ａｌｉｇｎｍｅｎｔ　Ｓｅａｒｃｈ　Ｔｏｏｌ）等のアライメントサーチツールを用いて決定することができる。当業者は、アラインメントにおいて、比較配列の最大のアラインメントを得るために適切なパラメーターを決定することができる。ヌクレオチド配列の「配列同一性」についても、同様の方法によって決定することができる。 As used herein, the percentage of "sequence identity" of a comparative amino acid sequence relative to a reference amino acid sequence is expressed by aligning the sequences so that the identity between these two sequences is maximized, and if necessary Defined as the percentage of amino acid residues in a comparison sequence that are identical to amino acid residues in a reference sequence when gaps are introduced in one or both of the two sequences. At this time, conservative substitutions are not considered part of sequence identity. Sequence identity can be determined by using publicly available computer software, for example, using an alignment search tool such as BLAST (registered trademark) (Basic Local Alignment Search Tool). can decide. Those skilled in the art can determine appropriate parameters for maximal alignment of the comparison sequences in the alignment. "Sequence identity" of nucleotide sequences can also be determined by similar methods.
　上記のようにして得られる遺伝子組み換え微生物は、ジアミン生産のために、その生育及び／または維持に適した条件下で培養及び維持される。各種の宿主微生物細胞に由来する形質転換体のための好適な培地組成、培養条件および培養時間は、当業者により選択される。 The genetically modified microorganism obtained as described above is cultured and maintained under conditions suitable for its growth and/or maintenance for diamine production. Appropriate medium compositions, culture conditions and culture times for transformants derived from various host microbial cells are selected by those skilled in the art.
　したがって、本発明の第二の側面は、先述の組換え微生物を培養することを含む、ジアミン化合物の製造方法に関する。具体的には、当該製造方法は、先述の組換え微生物を培養して、当該組み換え微生物の培養物および／または培養物の抽出物を得る培養工程を含む。 Therefore, the second aspect of the present invention relates to a method for producing a diamine compound, which includes culturing the aforementioned recombinant microorganism. Specifically, the production method includes a culturing step of culturing the aforementioned recombinant microorganism to obtain a culture of the recombinant microorganism and/or an extract of the culture.
　培養工程で用いる培地は、上記発明Ａにおいて説明したとおりである。 The medium used in the culture process is as explained in Invention A above.
　バイオマス由来の原料を用いた場合、製品であるジアミンは、発明Ａにおいて説明した測定法により、合成原料と明確に区別することができる。 When biomass-derived raw materials are used, the product diamine can be clearly distinguished from synthetic raw materials by the measurement method described in Invention A.
　上記培地に関し、セルロースおよび多糖類などの炭素源を宿主微生物が資化できない場合は、当該宿主微生物に発明Ａにおいて説明した遺伝子工学的手法を施すことで、炭素源を使用したジアミン生産に適応させることができる。 Regarding the above medium, if the host microorganism cannot assimilate carbon sources such as cellulose and polysaccharides, the host microorganism is adapted to diamine production using the carbon source by applying the genetic engineering technique described in Invention A. be able to.
　培養形式および培養条件は、発明Ａにおいて説明したとおりである。 The culture format and culture conditions are as explained in Invention A.
　本発明にかかる製造方法は、好ましくは、前記培養物および／または前記培養物の抽出物を、基質化合物と混合して混合液を得る混合工程をさらに含む。 The production method according to the present invention preferably further includes a mixing step of mixing the culture and/or the culture extract with a substrate compound to obtain a mixed solution.
　前記培養物および／または混合液中には、反応の結果、目的化合物であるジアミン化合物が生成する。よって、より好ましい態様において、本発明にかかる製造方法は、培養物または／および混合液から、ジアミン化合物を回収する回収工程をさらに含む。 A diamine compound, which is the target compound, is produced in the culture and/or mixed solution as a result of the reaction. Therefore, in a more preferred embodiment, the production method according to the present invention further includes a recovery step of recovering the diamine compound from the culture and/or the mixed solution.
　本発明にかかる製造方法は、発明Ａにおいて説明した工程から選択される１または複数を含んでもよい。 The manufacturing method according to the present invention may include one or more selected from the steps described in Invention A.
　上記のとおり、本発明は、ジアミン生産能を有する好塩性および／または好アルカリ性の組換え微生物であって、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成するＮ－アセチル化酵素を抑制する１以上の遺伝子改変を含む。本発明の組換え微生物の培養においては、副生物であるＮ－アセチルジアミン化合物の生成を抑制することができる。本発明の組換え微生物はジアミン生産能を有するものであり、副生物の生成を抑制しつつ、ジアミンを生成することができる。そのため、目的化合物であるジアミンを効率的に得ることができる。さらに、本発明にかかる組換え微生物は、工業的規模でのジアミン化合物生産への応用も期待される。 As described above, the present invention provides a halophilic and/or alkalophilic recombinant microorganism capable of producing diamine, which is an N-acetyltransferase that N-acetylates a diamine compound to produce an N-acetyldiamine compound. contains one or more genetic modifications that suppress In culturing the recombinant microorganism of the present invention, the production of N-acetyldiamine compounds, which are by-products, can be suppressed. The recombinant microorganism of the present invention has diamine-producing ability and can produce diamine while suppressing the production of by-products. Therefore, the target compound diamine can be efficiently obtained. Furthermore, the recombinant microorganism according to the present invention is also expected to be applied to the production of diamine compounds on an industrial scale.
　また、本発明にかかる組換え微生物が好アルカリ性である場合、当該微生物をジアミン生産に用いることで、酸溶液添加によるｐＨ調整を行う必要がなく、ジアミン製造プロセスの煩雑化を回避することが可能である。また、本発明にかかる組換え微生物が好塩性である場合、塩が含まれる培養液中でも生育可能であり、培養液または混合液（反応液）からジアミンを含む相を分離する分離工程で添加する塩を含む廃液を当該微生物の培養に再利用することができるため、排水処理コストを低減することができる。 In addition, when the recombinant microorganism according to the present invention is alkalophilic, by using the microorganism for diamine production, there is no need to adjust the pH by adding an acid solution, and it is possible to avoid complication of the diamine production process. is. In addition, when the recombinant microorganism according to the present invention is halophilic, it can grow even in a culture solution containing salt, and is added in the separation step of separating a phase containing diamine from the culture solution or mixed solution (reaction solution). Since the waste liquid containing the salt can be reused for culturing the microorganism, the waste water treatment cost can be reduced.
　以上、本発明を実施するための形態を例示したが、上記実施形態はあくまでも例として示されるものであり、発明の範囲を限定することを意図するものではない。上記実施形態は、その他の様々な形態で実施されることが可能であり、発明の要旨を逸脱しない範囲で、種々の省略、置換、変更を行うことができる。 Although the embodiments for carrying out the present invention have been illustrated above, the above embodiments are merely examples and are not intended to limit the scope of the invention. The above embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the scope of the invention.
＜１＞発明Ａ
　以下、本発明Ａを実施例に基づいて説明するが、本発明Ａはこれらの実施例に限定されるものではない。 <1> Invention A
The present invention A will be described below based on examples, but the present invention A is not limited to these examples.
　本実施例に示す全てのＰＣＲは、ＰｒｉｍｅＳＴＡＲ　Ｍａｘ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（製品名、タカラバイオ製）を用いて実施した。バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｅｕｄｏｆｉｒｍｕｓ）、バチルス・ハロデュランス（Ｂａｃｉｌｌｕｓ　ｈａｌｏｄｕｒａｎｓ）、バチルス・マーマエンシス（Ｂａｃｉｌｌｕｓ　ｍａｒｍａｒｅｎｓｉｓ）の形質転換は、エレクトロポレーション法を用いた。エレクトロポレーション法では、１μｌのプラスミドＤＮＡを６０μｌのコンピテントセルと共に入れた幅０．１ｃｍのキュベットをｇｅｎｅ　ｐｕｌｓａｒ（Ｂｉｏ－Ｒａｄ製）に装着して、電圧２．５ｋＶ・抵抗２００Ω・キャパシタンス２５μＦのパルスをキュベットに負荷した。３７℃にて３時間復帰培養した後、クロラムフェニコール１０μｇ／ｍＬを含む１８１培地に塗布し、形質転換体を取得した。１８１培地組成を表Ａ－１に示す。 All PCRs shown in this example were performed using PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio). Transformation of Bacillus pseudofirmus, Bacillus halodurans, and Bacillus marmarensis used the electroporation method. In the electroporation method, a cuvette with a width of 0.1 cm containing 1 μl of plasmid DNA and 60 μl of competent cells was attached to a gene pulsar (manufactured by Bio-Rad) and subjected to a voltage of 2.5 kV, a resistance of 200 Ω, and a capacitance of 25 μF. The pulse was loaded into the cuvette. After reincubation at 37° C. for 3 hours, the cells were plated on 181 medium containing 10 μg/mL chloramphenicol to obtain transformants. 181 medium composition is shown in Table A-1.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
実施例Ａ１：バチルス・シュードフィラマス用ｃａｄＡ遺伝子発現プラスミドの構築、および形質転換体の取得
（実施例Ａ１－ａ）プロモーター領域のクローニング
　バチルス・シュードフィラマスＯＦ４株（ＪＣＭ１７０５５株、本株は文部科学省ナショナルリソースプロジェクトを介して、理研ＢＲＣから提供された）を１８１培地中（２ｍｌ）で３７℃にて振盪培養した。培養終了後、培養液から菌体を回収し、Ｎｕｃｌｅｏ　Ｓｐｉｎ　Ｔｉｓｓｕｅ（製品名、ＭＡＣＨＥＲＥＹ－ＮＡＧＥＬ社製）を使用してゲノムＤＮＡを抽出した。バチルス・シュードフィラマスのｒｐｏＤ遺伝子のプロモーター領域（配列番号５）を、配列番号６、配列番号７に記載の配列を有するプライマーセットによりＰＣＲ増幅した（断片１）。反応条件は９８℃（１０ｓｅｃ），５５℃（５ｓｅｃ），７２℃（３０ｓｅｃ），３０ｃｙｃｌｅとした。
　プラスミドｐＡＬ３５１（ＮＩＴＥ　Ｐ－０２９１８として、独立行政法人製品評価技術基盤機構　バイオテクノロジーセンター　特許微生物寄託センター（ＮＰＭＤ）（住所：千葉県木更津市かずさ鎌足２－５－８　１２２号室）に２０１９年３月１８日付けで寄託され、その後、２０２０年７月２８日に、ブダペスト条約に基づく国際寄託へ移管されて、受託番号ＮＩＴＥ　ＢＰ－０２９１８が付与されている。）を、配列番号８および配列番号９に示すプライマーセットによりＰＣＲ増幅した（断片２）。反応条件は９８℃（１０ｓｅｃ），５５℃（５ｓｅｃ），７２℃（３０ｓｅｃ），３０ｃｙｃｌｅとした。断片２をベクター側とし、インサートとして断片１をライゲーションしｐＡＬＰ０１を構築した。 Example A1: Construction of cadA gene expression plasmid for Bacillus pseudofilamus and acquisition of transformant (Example A1-a) Cloning of promoter region Bacillus pseudofilamus OF4 strain (JCM17055 strain, this strain is (provided by RIKEN BRC through the Ministry of National Resources Project) was shake-cultured in 181 medium (2 ml) at 37°C. After the culture was completed, the cells were recovered from the culture medium, and genomic DNA was extracted using Nucleo Spin Tissue (product name, MACHEREY-NAGEL). The promoter region (SEQ ID NO: 5) of the rpoD gene of Bacillus pseudofilamus was PCR-amplified (fragment 1) with a primer set having the sequences shown in SEQ ID NOS: 6 and 7. The reaction conditions were 98° C. (10 sec), 55° C. (5 sec), 72° C. (30 sec), and 30 cycles.
Plasmid pAL351 (as NITE P-02918, National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary Center (NPMD) (Address: 2-5-8 Kazusa Kamatari, Chiba Prefecture, Room 122) in March 2019 18, and was subsequently transferred to an international deposit under the Budapest Treaty on July 28, 2020, with accession number NITE BP-02918.), SEQ ID NO: 8 and SEQ ID NO: 9. PCR amplification was performed using the primer set shown in (Fragment 2). The reaction conditions were 98° C. (10 sec), 55° C. (5 sec), 72° C. (30 sec), and 30 cycles. With fragment 2 on the vector side, fragment 1 was ligated as an insert to construct pALP01.
（実施例Ａ１－ｂ）ｃａｄＡ遺伝子のクローニング
　大腸菌Ｗ３１１０株（ＮＢＲＣ１２７１３）をＬＢ培地中（２ｍＬ）で３７度にて振とう培養した。培養終了後、培養液から菌体を回収し、Ｎｕｃｌｅｏ　Ｓｐｉｎ　Ｔｉｓｓｕｅを使用してゲノムＤＮＡを抽出した。抽出したゲノムＤＮＡを鋳型に、配列番号１０、配列番号１１に記載の配列を有するプライマーセットにてＰＣＲ増幅した。反応条件は９８℃（１０ｓｅｃ），５５℃（５ｓｅｃ），７２℃（３０ｓｅｃ），３０ｃｙｃｌｅとした。増幅断片をｐＡＬＰ０１のプロモーター配列の下流にライゲーションしてｐＡＬ３２８を構築した。 (Example A1-b) Cloning of cadA gene Escherichia coli strain W3110 (NBRC12713) was shake-cultured in LB medium (2 mL) at 37°C. After completion of the culture, the cells were recovered from the culture medium, and genomic DNA was extracted using Nucleo Spin Tissue. Using the extracted genomic DNA as a template, PCR amplification was performed with a primer set having the sequences shown in SEQ ID NO: 10 and SEQ ID NO: 11. The reaction conditions were 98° C. (10 sec), 55° C. (5 sec), 72° C. (30 sec), and 30 cycles. The amplified fragment was ligated downstream of the promoter sequence of pALP01 to construct pAL328.
（実施例Ａ１－ｃ）形質転換体の取得
　実施例Ａ１－ｂで構築したｐＡＬ３２８を、
・バチルス・シュードフィラマスＯＦ４株（ＪＣＭ１７０５５株、本株は文部科学省ナショナルリソースプロジェクトを介して、理研ＢＲＣから提供された）、
・バチルス・ハロデュランスＣ－１２５株（ＪＣＭ９１５３株、本株は文部科学省ナショナルリソースプロジェクトを介して、理研ＢＲＣから提供された）、
・バチルス・マーマエンシス　ＧＭＢＥ７２株（ＪＣＭ１５７１９株、本株は文部科学省ナショナルリソースプロジェクトを介して、理研ＢＲＣから提供された）
に形質転換し、それぞれ、
・ＡＫＲＭ－１株、
・ＡＫＲＭ－２株、
・ＡＫＲＭ－３株
を取得した。 (Example A1-c) Acquisition of transformant pAL328 constructed in Example A1-b,
- Bacillus pseudofilamus OF4 strain (JCM17055 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project),
- Bacillus halodurans C-125 strain (JCM9153 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project),
- Bacillus marmaensis GMBE72 strain (JCM15719 strain, this strain was provided by RIKEN BRC via the Ministry of Education, Culture, Sports, Science and Technology National Resource Project)
, respectively,
・ AKRM-1 strain,
・ AKRM-2 strain,
・The AKRM-3 strain was acquired.
　一方、対照の形質転換ではｃａｄＡ遺伝子を含まないプラスミドｐＡＬＰ０１を、
・バチルス・シュードフィラマスＯＦ４株（ＪＣＭ１７０５５株、本株は文部科学省ナショナルリソースプロジェクトを介して、理研ＢＲＣから提供された）、
・バチルス・ハロデュランスＣ－１２５株（ＪＣＭ９１５３株、本株は文部科学省ナショナルリソースプロジェクトを介して、理研ＢＲＣから提供された）、
・バチルス・マーマエンシス　ＧＭＢＥ７２株（ＪＣＭ１５７１９株、本株は文部科学省ナショナルリソースプロジェクトを介して、理研ＢＲＣから提供された）
に形質転換し、それぞれ、
・ＡＫＲＭ－４株、
・ＡＫＲＭ－５株、
・ＡＫＲＭ－６株
を取得した。 On the other hand, in the control transformation, the plasmid pALP01, which does not contain the cadA gene,
- Bacillus pseudofilamus OF4 strain (JCM17055 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project),
- Bacillus halodurans C-125 strain (JCM9153 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project),
- Bacillus marmaensis GMBE72 strain (JCM15719 strain, this strain was provided by RIKEN BRC via the Ministry of Education, Culture, Sports, Science and Technology National Resource Project)
, respectively,
・ AKRM-4 strain,
・ AKRM-5 strain,
・The AKRM-6 strain was acquired.
　さらにバチルス・シュードフィラマスＯＦ４株についてはｃａｄＡ遺伝子発現カセットを染色体上に有する形質転換体も取得した。本株バチルス・シュードフィラマスＡＫＡＬ－００１株は、ＮＩＴＥ　Ｐ－０２９２０として、独立行政法人製品評価技術基盤機構　バイオテクノロジーセンター　特許微生物寄託センター（ＮＰＭＤ）（住所：千葉県木更津市かずさ鎌足２－５－８　１２２号室）に２０１９年３月１８日付けで寄託され、その後、２０２０年７月２８日に、ブダペスト条約に基づく国際寄託へ移管されて、受託番号ＮＩＴＥ　ＢＰ－０２９２０が付与されている。 Furthermore, for the Bacillus pseudofilamus OF4 strain, a transformant with a cadA gene expression cassette on the chromosome was also obtained. This strain, Bacillus pseudofilamus AKAL-001, has been designated as NITE P-02920, National Institute of Technology and Evaluation, National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary Center (NPMD) (Address: 2-5 Kazusa Kamatari, Kisarazu City, Chiba Prefecture). -8 Room 122) on March 18, 2019, and then on July 28, 2020, it was transferred to an international deposit under the Budapest Treaty and was given accession number NITE BP-02920.
　上記配列を以下に示す。 The above array is shown below.
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
実施例Ａ２：構築した菌株によるカダベリン生産(フラスコ培養)
　得られた各々の形質転換体を、１８１培地プレート上で、３７℃、２日間培養して、コロニーを形成させた。１８１培地２ｍＬを１４ｍＬ容の試験管に入れ、上記プレートからコロニーを白金耳で植菌し、３７℃で、１８０ｒｐｍで培養を行い、十分な濁度を得るまで培養し、これを本培養のための前培養液とした。 Example A2: Cadaverine production by constructed strain (flask culture)
Each transformant obtained was cultured on a 181 medium plate at 37° C. for 2 days to form colonies. Put 2 mL of 181 medium in a 14 mL test tube, inoculate a colony from the plate with a platinum loop, culture at 37 ° C. and 180 rpm, culture until sufficient turbidity is obtained, and use this for the main culture It was used as a pre-culture solution.
　１５０ｍＬ容の三角フラスコに、ＢＳ培地（組成を表Ａ－２に示す）を３０ｍＬ入れ、０．３ｍＬの前培養液を添加し本培養（カダベリン生産試験）を行った。培養条件は３７℃で、１８０ｒｐｍとした。なお、ＡＫＲＭ－１～ＡＫＲＭ－６株培養時には、前培養、本培養共に培地には１０ｍｇ／Ｌのクロラムフェニコールを添加した。 30 mL of BS medium (composition shown in Table A-2) was placed in a 150 mL Erlenmeyer flask, 0.3 mL of pre-culture solution was added, and main culture (cadaverine production test) was performed. The culture conditions were 37° C. and 180 rpm. In culturing the AKRM-1 to AKRM-6 strains, 10 mg/L of chloramphenicol was added to the medium for both preculture and main culture.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　上記の培養液を、１０，０００ｇ×３分間遠心分離して上清を回収し、培養上清のカダベリン濃度を測定した。具体的には、ＣＧ－１９（ガードカラム）、ＣＳ－１９（分析カラム）（いずれも商品名、サーモフィッシャーサイエンティフィク株式会社製）を連結してイオンクロマト分析（検出器：電気伝導度、カラム温度：３０℃、流速：０．３５ｍＬ／ｍｉｎ、移動相：８ｍＭメタンスルホン酸水溶液→７０ｍＭ　メタンスルホン酸水溶液のグラジエント）を行うことで、培養上清中のカダベリン濃度を定量した。カダベリン濃度に関し、本発明の形質転換体（ＡＫＲＭ－１株、ＡＫＲＭ－２株、ＡＫＲＭ－３株、ＡＫＡＬ－００１株）と対照株（ＡＫＲＭ－４株、ＡＫＲＭ－５株、ＡＫＲＭ－６株）との比較結果を表Ａ－３に示した。 The above culture solution was centrifuged at 10,000 g for 3 minutes, the supernatant was collected, and the cadaverine concentration of the culture supernatant was measured. Specifically, CG-19 (guard column) and CS-19 (analytical column) (both trade names, manufactured by Thermo Fisher Scientific Co., Ltd.) are connected to perform ion chromatography analysis (detector: electrical conductivity, Column temperature: 30°C, flow rate: 0.35 mL/min, mobile phase: 8 mM methanesulfonic acid aqueous solution → 70 mM methanesulfonic acid aqueous gradient) was performed to quantify the cadaverine concentration in the culture supernatant. Regarding the cadaverine concentration, the transformants of the present invention (AKRM-1 strain, AKRM-2 strain, AKRM-3 strain, AKAL-001 strain) and control strains (AKRM-4 strain, AKRM-5 strain, AKRM-6 strain) The results of comparison with are shown in Table A-3.
　対照株（ＡＫＲＭ－４株、ＡＫＲＭ－５株、ＡＫＲＭ－６株）に比べて、本発明の形質転換体（ＡＫＲＭ－１株、ＡＫＲＭ－２株、ＡＫＲＭ－３株、ＡＫＡＬ－００１株）は、培養時間の経過に伴い、より多くのカダベリンを生産した。 Compared to the control strains (AKRM-4 strain, AKRM-5 strain, AKRM-6 strain), the transformants of the present invention (AKRM-1 strain, AKRM-2 strain, AKRM-3 strain, AKAL-001 strain) , produced more cadaverine with the passage of culture time.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
実施例Ａ３：構築した菌株によるカダベリン生産（ファーメンター培養）
　ＡＫＲＭ－１株、ＡＫＲＭ－４株、およびＡＫＡＬ－００１株を、１８１培地プレート上で、３７℃、２日間培養して、コロニーを形成させた。１８１培地１００ｍＬを５００ｍＬ容の三角フラスコに入れ、上記プレートからコロニーを白金耳で植菌し、３７℃で、１８０ｒｐｍで培養を行い、十分な濁度を得るまで培養し、これを本培養のための前培養液とした。 Example A3: Cadaverine production by constructed strain (fermenter culture)
AKRM-1 strain, AKRM-4 strain, and AKAL-001 strain were cultured on a 181 medium plate at 37° C. for 2 days to form colonies. Put 100 mL of 181 medium in a 500 mL Erlenmeyer flask, inoculate a colony from the plate with a platinum loop, culture at 37 ° C. and 180 rpm until sufficient turbidity is obtained. It was used as a pre-culture solution.
　１０Ｌ容のジャー培養装置（機種名：ＭＤＬ－６Ｃ、丸菱バイオエンジ社製）に２０ｇ／Ｌグルコースを含むＢＳジャー培地（表Ａ－４に示す）を入れ、１００ｍＬの前培養液を添加し本培養（カダベリン生産試験）を行った。なおＡＫＲＭ－１株、ＡＫＲＭ－４株培養時には、前培養、本培養共に培地には１０ｍｇ／Ｌのクロラムフェニコールを添加した。培養条件は、培養温度：３７℃、培養ｐＨ：７．５、アルカリ添加：２８％アンモニア水、攪拌速度：７００ｒｐｍ、通気速度：０．１ｖｖｍとした。また、培養途中からフィード培地（組成を表Ａ－５に示す）を培地中グルコース濃度が０～５ｇ／Ｌとなるように逐次添加した。培養中に経時的にサンプリングを行い、培養上清中のカダベリン濃度を定量した。 A BS jar medium containing 20 g/L glucose (shown in Table A-4) was placed in a 10 L jar culture apparatus (model name: MDL-6C, manufactured by Marubishi Bioengineering Co., Ltd.), and 100 mL of the preculture solution was added. A main culture (cadaverine production test) was performed. In culturing the AKRM-1 and AKRM-4 strains, 10 mg/L of chloramphenicol was added to the medium for both preculture and main culture. The culture conditions were culture temperature: 37° C., culture pH: 7.5, alkali addition: 28% ammonia water, stirring speed: 700 rpm, aeration speed: 0.1 vvm. In addition, a feed medium (composition shown in Table A-5) was sequentially added during the culture so that the glucose concentration in the medium was 0 to 5 g/L. Sampling was performed over time during the culture, and the cadaverine concentration in the culture supernatant was quantified.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　結果を表Ａ－６に示す。ＡＫＲＭ－４株はカダベリンを生産せず、プラスミド上でｃａｄＡ遺伝子を発現するＡＫＲＭ－１株ではカダベリンの濃度が増加せず、染色体上でｃａｄＡ遺伝子を発現するＡＫＡＬ－００１株では培養時間の経過に伴いカダベリン濃度が増加した。 The results are shown in Table A-6. The AKRM-4 strain does not produce cadaverine, the AKRM-1 strain that expresses the cadA gene on the plasmid does not increase the concentration of cadaverine, and the AKAL-001 strain that expresses the cadA gene on the chromosome decreases the cadaverine concentration with the passage of culture time. Cadaverine concentration increased accordingly.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
実施例Ａ４：硫酸ナトリウム濃度を上げたときのカダベリン生産(フラスコ培養)
　ＡＫＡＬ－００１株を、１８１培地プレート上で、３７℃、２日間培養して、コロニーを形成させた。１８１培地２ｍＬを１４ｍＬ容の試験管に入れ、上記プレートからコロニーを白金耳で植菌し、３７℃で、１８０ｒｐｍで培養を行い、十分な濁度を得るまで培養し、これを本培養のための前培養液とした。 Example A4: Cadaverine production with increasing sodium sulfate concentration (flask culture)
The AKAL-001 strain was cultured on a 181 medium plate at 37° C. for 2 days to form colonies. Put 2 mL of 181 medium in a 14 mL test tube, inoculate a colony from the plate with a platinum loop, culture at 37 ° C. and 180 rpm, culture until sufficient turbidity is obtained, and use this for the main culture It was used as a pre-culture solution.
　１５０ｍＬ容の三角フラスコに、ＢＳ培地（表Ａ－２に示す）３０ｍＬを２本用意した。１本には表Ａ－２に示す通り５０ｇ／Ｌの硫酸ナトリウムを添加し、もう１本には１００ｇ／Ｌの硫酸ナトリウムを添加した。０．３ｍＬの前培養液を添加し本培養（カダベリン生産試験）を行った。培養条件は３７℃で、１８０ｒｐｍとした。 Two 30 mL bottles of BS medium (shown in Table A-2) were prepared in a 150 mL Erlenmeyer flask. One was added with 50 g/L sodium sulfate as shown in Table A-2, and the other was added with 100 g/L sodium sulfate. A main culture (cadaverine production test) was performed by adding 0.3 mL of the preculture solution. The culture conditions were 37° C. and 180 rpm.
　上記の培養液を、１０，０００ｇ×３分間遠心分離して上清を回収し、実施例Ａ２に記載の方法にて培養上清のカダベリン濃度を測定した結果を表Ａ－７に示した。ＡＫＡＬ－００１株は、１００ｇ／Ｌの高濃度の硫酸ナトリウム存在下でもカダベリンを産生し、培養時間の経過に伴いカダベリン濃度の増加を示した。 The above culture solution was centrifuged at 10,000 g for 3 minutes, the supernatant was collected, and the cadaverine concentration of the culture supernatant was measured by the method described in Example A2, and the results are shown in Table A-7. The AKAL-001 strain produced cadaverine even in the presence of sodium sulfate at a high concentration of 100 g/L, and showed an increase in cadaverine concentration with the passage of culture time.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
実施例Ａ５：各濃度の塩化合物添加時の相分離性
　分離確認用模擬液 (組成を表Ａ－８に示す)に、カダベリン(１，５－ジアミノペンタン、富士フィルムワコーケミカル製)と塩化ナトリウム、硫酸ナトリウム、炭酸ナトリウムとを種々の濃度で添加して、模擬液を５ｍＬずつ作製した。ボルテックスミキサーで混合後、室温で１０分静置した後の相分離の様子と水相、カダベリン相の液量を確認した。また、水相に含まれるカダベリン濃度を測定し、水相に残存するカダベリン量を算出し、水相からカダベリン相への移行率を算出した。 Example A5: Cadaverine (1,5-diaminopentane, manufactured by Fuji Film Wako Chemical) and sodium chloride were added to a simulated liquid for confirming phase separation when salt compounds were added at various concentrations (the composition is shown in Table A-8). , sodium sulfate, and sodium carbonate at various concentrations to prepare 5 mL of simulated solutions. After mixing with a vortex mixer, the mixture was allowed to stand at room temperature for 10 minutes, and the state of phase separation and the amounts of the aqueous phase and the cadaverine phase were confirmed. Also, the concentration of cadaverine contained in the aqueous phase was measured, the amount of cadaverine remaining in the aqueous phase was calculated, and the transfer rate from the aqueous phase to the cadaverine phase was calculated.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
　最終的に作製した溶液組成と分離結果を表Ａ－９に示した。表中、相分離欄において、「×」は相分離が発生しないこと、「〇」は目視確認において、形成したジアミン相が総液量の１０％未満（例えば総液量が５ｍＬであればジアミン相が０．５ｍＬ未満）であること、「◎」は目視確認において、形成したジアミン相が総液量の１０％以上発生していることを示す。相分離に関する本評価基準は、以降の表についても同様に適用される。試験の結果、各無機塩を高濃度添加した条件において、ジアミン相分離が生ずることを確認した。 Table A-9 shows the final solution composition and separation results. In the table, in the phase separation column, "x" indicates that phase separation does not occur, and "○" indicates that the formed diamine phase is less than 10% of the total liquid volume (for example, if the total liquid volume is 5 mL, diamine phase is less than 0.5 mL), and "⊚" indicates that the diamine phase formed is 10% or more of the total liquid volume by visual confirmation. This evaluation criterion for phase separation applies to the following tables as well. As a result of the test, it was confirmed that the diamine phase separation occurred under the condition that each inorganic salt was added at a high concentration.
Figure JPOXMLDOC01-appb-T000013
Figure JPOXMLDOC01-appb-T000013
実施例Ａ６：カダベリンおよびヘキサメチレンジアミン（ＨＭＤＡ）の相分離性
　分離確認用模擬液（組成を表Ａ－８に示す）に、硫酸ナトリウムとカダベリン(１，５－ジアミノペンタン、富士フィルムワコーケミカル製)またはヘキサメチレンジアミン(富士フィルム和光純薬製)とを種々の濃度で添加して、模擬液を５ｍＬずつ作製した。ボルテックスミキサーで混合後、室温で１０分静置した後の相分離の様子と水相、カダベリン相の液量を確認した。また、水相に含まれるカダベリン濃度を測定し、水相に残存するカダベリン量を算出し、水相からカダベリン相への移行率を算出した。 Example A6: Sodium sulfate and cadaverine (1,5-diaminopentane, manufactured by Fujifilm Wako Chemical Co., Ltd.) were added to a simulated liquid for confirming phase separation of cadaverine and hexamethylenediamine (HMDA) (the composition is shown in Table A-8). ) or hexamethylenediamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at various concentrations to prepare 5 mL of simulated solutions. After mixing with a vortex mixer, the mixture was allowed to stand at room temperature for 10 minutes, and the state of phase separation and the amounts of the aqueous phase and the cadaverine phase were confirmed. Also, the concentration of cadaverine contained in the aqueous phase was measured, the amount of cadaverine remaining in the aqueous phase was calculated, and the transfer rate from the aqueous phase to the cadaverine phase was calculated.
　最終的に作製した溶液組成と分離結果を表Ａ－１０に示した。高濃度の硫酸ナトリウム存在下ではカダベリン、ヘキサメチレンジアミン（ＨＭＤＡ）が共に相分離することを確認した。 Table A-10 shows the final solution composition and separation results. It was confirmed that both cadaverine and hexamethylenediamine (HMDA) undergo phase separation in the presence of high-concentration sodium sulfate.
Figure JPOXMLDOC01-appb-T000014
Figure JPOXMLDOC01-appb-T000014
実施例Ａ７：各濃度の炭酸ナトリウム添加時の溶媒抽出性(カダベリン)
　分離確認用模擬液（組成を表Ａ－８に示す）に、各濃度の炭酸ナトリウムとカダベリン(１，５－ジアミノペンタン、富士フィルムワコーケミカル製)とを添加して、模擬液を５ｍＬずつ作製した。模擬液に、等量（５ｍＬ）のｎ－ブタノールを加え、ボルテックスミキサーで混合後、室温で１０分静置した。水相に含まれるカダベリン濃度を測定し、水相に残存するカダベリン量を算出し、水相から溶媒相への移行率を算出した。 Example A7: Solvent extractability (cadaverine) when adding sodium carbonate at each concentration
Each concentration of sodium carbonate and cadaverine (1,5-diaminopentane, manufactured by Fuji Film Wako Chemical) was added to the simulated solution for confirmation of separation (composition shown in Table A-8) to prepare 5 mL of simulated solution. did. An equal amount (5 mL) of n-butanol was added to the simulated solution, mixed with a vortex mixer, and allowed to stand at room temperature for 10 minutes. The concentration of cadaverine contained in the aqueous phase was measured, the amount of cadaverine remaining in the aqueous phase was calculated, and the migration rate from the aqueous phase to the solvent phase was calculated.
　最終的に作製した溶液組成と分離結果を表Ａ－１１に示した。炭酸ナトリウムの濃度上昇に伴い、溶媒への抽出効率が向上することを確認した。 Table A-11 shows the final solution composition and separation results. It was confirmed that the extraction efficiency into the solvent improved as the concentration of sodium carbonate increased.
Figure JPOXMLDOC01-appb-T000015
Figure JPOXMLDOC01-appb-T000015
実施例Ａ８：各濃度の硫酸ナトリウム添加時の溶媒抽出性(ヘキサメチレンジアミン)
　分離確認用模擬液 (組成を表Ａ－８に示す)に、各濃度の硫酸ナトリウムとヘキサメチレンジアミンとを添加して、模擬液を５ｍＬずつ作製した。模擬液に、等量（５ｍＬ）のヘキサンまたは２－エチル－１－ヘキサノールを加え、ボルテックスミキサーで混合後、室温で１０分静置した。溶媒相に含まれるヘキサメチレンジアミン濃度を測定し、水相から溶媒相への移行率を算出した。 Example A8: Solvent extractability (hexamethylenediamine) when sodium sulfate is added at various concentrations
Each concentration of sodium sulfate and hexamethylenediamine was added to a simulated liquid for confirmation of separation (composition shown in Table A-8) to prepare 5 mL of simulated liquid. An equal amount (5 mL) of hexane or 2-ethyl-1-hexanol was added to the simulated solution, mixed with a vortex mixer, and allowed to stand at room temperature for 10 minutes. The concentration of hexamethylenediamine contained in the solvent phase was measured, and the migration rate from the aqueous phase to the solvent phase was calculated.
　最終的に作製した溶液組成と分離結果を表Ａ－１２に示した。硫酸ナトリウムの濃度上昇に伴い、２－エチル－１－ヘキサノールへの抽出効率が向上すること、ｎ－ヘキサンへの抽出効率は非常に低いことを確認した。 Table A-12 shows the final solution composition and separation results. It was confirmed that the extraction efficiency into 2-ethyl-1-hexanol improved as the concentration of sodium sulfate increased, while the extraction efficiency into n-hexane was very low.
Figure JPOXMLDOC01-appb-T000016
Figure JPOXMLDOC01-appb-T000016
実施例Ａ９：ジアミン添加培養液での相分離性
　実施例Ａ４記載のフラスコ培養の培養上清３０ｍＬを０．２２μｍのフィルターでろ過し、カダベリン(１，５－ジアミノペンタン、富士フィルムワコーケミカル製)またはヘキサメチレンジアミン(富士フィルム和光純薬製)を各濃度になるように添加し、エバポレーターにて２倍濃縮した（収量１５ｍＬ）。本濃縮液をボルテックスミキサーで混合後、室温で１０分静置した後の相分離の様子と水相、カダベリン相の液量を確認した。また、水相に含まれるカダベリン濃度を測定し、水相に残存するカダベリン量を算出し、水相からカダベリン相への移行率を算出した。最終的に作製した溶液組成と分離結果を表Ａ－１３に示した。培養液中に高濃度の硫酸ナトリウムが存在するとカダベリン、ヘキサメチレンジアミン共に相分離することを確認した。 Example A9: Phase separation in diamine-added culture medium 30 mL of the culture supernatant of the flask culture described in Example A4 was filtered through a 0.22 μm filter, and cadaverine (1,5-diaminopentane, manufactured by Fujifilm Wako Chemical ) or hexamethylenediamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each concentration, and concentrated twice with an evaporator (yield: 15 mL). After mixing the concentrate with a vortex mixer, the mixture was allowed to stand at room temperature for 10 minutes, and the state of phase separation and the amounts of the aqueous phase and the cadaverine phase were confirmed. Also, the concentration of cadaverine contained in the aqueous phase was measured, the amount of cadaverine remaining in the aqueous phase was calculated, and the transfer rate from the aqueous phase to the cadaverine phase was calculated. Table A-13 shows the finally prepared solution composition and separation results. It was confirmed that both cadaverine and hexamethylenediamine undergo phase separation when high concentrations of sodium sulfate are present in the culture medium.
Figure JPOXMLDOC01-appb-T000017
Figure JPOXMLDOC01-appb-T000017
実施例Ａ１０：相分離後の水相を再利用した培養
　実施例Ａ３記載のＡＫＡＬ－００１株のファーメンター培養液（培養６３時間）１００ｍＬを回収し、遠心分離し上清を回収した。培養上清１００ｍＬを０．２２μｍのフィルターでろ過し、エバポレーターにて２倍濃縮した（収量５０ｍＬ）。本濃縮液にヘキサメチレンジアミン(富士フィルム和光純薬製)を終濃度１５０ｇ／Ｌになるように添加した。ボルテックスミキサーで混合後、室温で１０分静置した後に相分離の形成を確認した。水相は２０ｍＬであり、ヘキサメチレンジアミンの濃度は４８．５ｇ／Ｌであった。この水相をピペットで回収し、硫酸でｐＨを７．５に調整した後、蒸留水を３０ｍＬ加え、５０ｍＬにメスアップした。本液と新しいＢＳ培地（組成を表Ａ－１４に示す）とを、比率を変えて混合し、１５０ｍＬ容の三角フラスコに用意した。 Example A10: Culture Reusing the Aqueous Phase After Phase Separation 100 mL of the fermenter culture medium (cultured for 63 hours) of the AKAL-001 strain described in Example A3 was recovered and centrifuged to recover the supernatant. 100 mL of the culture supernatant was filtered through a 0.22 μm filter and concentrated twice with an evaporator (yield: 50 mL). Hexamethylenediamine (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was added to the concentrate to a final concentration of 150 g/L. After mixing with a vortex mixer, the formation of phase separation was confirmed after standing at room temperature for 10 minutes. The aqueous phase was 20 mL and the concentration of hexamethylenediamine was 48.5 g/L. This aqueous phase was collected with a pipette, and after adjusting the pH to 7.5 with sulfuric acid, 30 mL of distilled water was added to make up to 50 mL. This solution and fresh BS medium (composition shown in Table A-14) were mixed at different ratios and prepared in a 150 mL Erlenmeyer flask.
Figure JPOXMLDOC01-appb-T000018
Figure JPOXMLDOC01-appb-T000018
　バチルス・シュードフィラマスＯＦ４株（ＪＣＭ１７０５５株、本株は文部科学省ナショナルリソースプロジェクトを介して、理研ＢＲＣから提供された）を、１８１培地プレート上で、３７℃、２日間培養して、コロニーを形成させた。１８１培地２ｍＬを１４ｍＬ容の試験管に入れ、上記プレートからコロニーを白金耳で植菌し、３７℃で、１８０ｒｐｍで培養を行い、十分な濁度を得るまで培養し、これを本培養のための前培養液とした。 Bacillus pseudofilamus OF4 strain (JCM17055 strain, this strain was provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project) was cultured on a 181 medium plate at 37 ° C. for 2 days to form colonies. formed. Put 2 mL of 181 medium in a 14 mL test tube, inoculate a colony from the plate with a platinum loop, culture at 37 ° C. and 180 rpm, culture until sufficient turbidity is obtained, and use this for the main culture It was used as a pre-culture solution.
　上記フラスコに用意した培地に０．１ｍＬの前培養液を添加し本培養を行った。培養条件は３７℃で、１８０ｒｐｍとした。培養４９時間後に培養液をサンプリングし、濁度（ＯＤ６００）測定を実施した。 　The main culture was performed by adding 0.1 mL of the pre-culture solution to the medium prepared in the flask. The culture conditions were 37° C. and 180 rpm. After 49 hours of culture, the culture solution was sampled and turbidity (OD600) was measured.
　結果を表Ａ－１５に示す。相分離後の水相を培地として再利用できることを確認した。 The results are shown in Table A-15. It was confirmed that the aqueous phase after phase separation could be reused as a medium.
Figure JPOXMLDOC01-appb-T000019
Figure JPOXMLDOC01-appb-T000019
　上記実施例記載の結果より、好塩性および好アルカリ性の両方の性質を持つ微生物を宿主として用いてジアミンを生産することが出来た。また、高塩濃度の炭酸ナトリウムまたは硫酸ナトリウム存在下ではジアミンが相分離することと、溶媒抽出時の抽出効率が向上することを確認した。 From the results described in the above example, it was possible to produce diamine using a microorganism that has both halophilic and alkalophilic properties as a host. In addition, it was confirmed that the diamine undergoes phase separation in the presence of sodium carbonate or sodium sulfate at a high salt concentration, and that the extraction efficiency during solvent extraction is improved.
　ジアミンを含む培養上清を濃縮することでジアミンおよび無機塩の濃度が上昇し、一層の分離効率向上が期待できる。また、本発明にかかる組換え微生物を利用することで、酸溶液添加によるｐＨ調整の工程を省略することができる。さらには、ジアミンを遊離させる際に発生する高濃度塩含有水を培地として再利用でき、排水処理コストの低減が可能となる。 By concentrating the culture supernatant containing diamine, the concentrations of diamine and inorganic salts increase, and further improvement in separation efficiency can be expected. Moreover, by using the recombinant microorganism according to the present invention, the step of adjusting pH by adding an acid solution can be omitted. Furthermore, the water containing high concentrations of salts generated when liberating the diamine can be reused as a medium, and the wastewater treatment cost can be reduced.
＜２＞発明Ｂ
　以下、本発明Ｂを実施例に基づいて説明するが、本発明Ｂはこれらの実施例に限定されるものではない。 <2> Invention B
Hereinafter, the present invention B will be described based on examples, but the present invention B is not limited to these examples.
　本実施例に示す全てのＰＣＲは、ＰｒｉｍｅＳＴＡＲ　Ｍａｘ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（製品名、タカラバイオ製）を用いて実施した。バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｅｕｄｏｆｉｒｍｕｓ）の形質転換は、エレクトロポレーション法を用いた。エレクトロポレーション法では、１μｌのプラスミドＤＮＡを６０μｌのコンピテントセルと共に入れた幅０．１ｃｍのキュベットをｇｅｎｅ　ｐｕｌｓａｒ（Ｂｉｏ－Ｒａｄ製）に装着して、電圧２．５ｋＶ、抵抗２００Ω、キャパシタンス２５μＦのパルスをキュベットに負荷した。３０℃にて３時間復帰培養した後、クロラムフェニコール１０μｇ／ｍＬを含む１８１培地に塗布し、形質転換体を取得した。１８１培地組成を表Ｂ－１に示す。 All PCRs shown in this example were performed using PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio). Transformation of Bacillus pseudofirmus used the electroporation method. In the electroporation method, a cuvette with a width of 0.1 cm containing 1 μl of plasmid DNA and 60 μl of competent cells was attached to a gene pulsar (manufactured by Bio-Rad), and a voltage of 2.5 kV, a resistance of 200 Ω, and a capacitance of 25 μF were applied. The pulse was loaded into the cuvette. After reincubation at 30° C. for 3 hours, the cells were plated on 181 medium containing 10 μg/mL chloramphenicol to obtain transformants. 181 medium composition is shown in Table B-1.
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000020
実施例Ｂ１：遺伝子破壊株の取得
　ＮＣＢＩのデータベース上に公開されているバチルス・シュードフィラマスＯＦ４株のアノテーション情報から、アセチルトランスフェラーゼをコードする３９遺伝子を、ジアミンのＮ－アセチル化酵素をコードする候補遺伝子として選別した。３９候補のうち、
・ＢｐＯＦ４＿１１２５５遺伝子（ＧｅｎＢａｎｋ：ＡＤＣ５０３０４）、
・ＢｐＯＦ４＿１６５１５遺伝子（ＧｅｎＢａｎｋ：ＡＤＣ５１３４８）、
・ＢｐＯＦ４＿１８３７５遺伝子（ＧｅｎＢａｎｋ：ＡＤＣ５１７１６）、
・ＢｐＯＦ４＿１６７２５遺伝子（ＧｅｎＢａｎｋ：ＡＤＣ５１３８８）、
・ＢｐＯＦ４＿１８１６０－６５遺伝子（ＧｅｎＢａｎｋ：ＡＤＣ５１６７３－４）、
・ＢｐＯＦ４＿１８５４５遺伝子（ＧｅｎＢａｎｋ：ＡＤＣ５１７５０）、
・ＢｐＯＦ４＿１９３８０遺伝子（ＧｅｎＢａｎｋ：ＡＤＣ５１９１５）、
・ＢｐＯＦ４＿００７５０遺伝子（ＧｅｎＢａｎｋ：ＡＤＣ４８２１９）、
・ＢｐＯＦ４＿０１９２５遺伝子（ＧｅｎＢａｎｋ：ＡＤＣ４８４５２）
の遺伝子破壊株を、相同組換えによる染色体への遺伝子操作で作製した。用いたプライマー配列を図５Ａおよび図５Ｂに示す。 Example B1: Acquisition of gene-disrupted strains From the annotation information of Bacillus pseudofilamus OF4 strain published on the NCBI database, 39 genes encoding acetyltransferase were identified as candidates encoding N-acetyltransferase of diamine. Selected as a gene. Of the 39 candidates,
- BpOF4_11255 gene (GenBank: ADC50304),
- BpOF4_16515 gene (GenBank: ADC51348),
- BpOF4_18375 gene (GenBank: ADC51716),
- BpOF4_16725 gene (GenBank: ADC51388),
- BpOF4_18160-65 gene (GenBank: ADC51673-4),
- BpOF4_18545 gene (GenBank: ADC51750),
- BpOF4_19380 gene (GenBank: ADC51915),
- BpOF4_00750 gene (GenBank: ADC48219),
・BpOF4_01925 gene (GenBank: ADC48452)
was generated by gene manipulation to the chromosome by homologous recombination. The primer sequences used are shown in Figures 5A and 5B.
　（実施例Ｂ１－ａ）遺伝子破壊プラスミドの作製
　バチルス・シュードフィラマスＯＦ４株（ＪＣＭ１７０５５株、本株は文部科学省ナショナルリソースプロジェクトを介して、理研ＢＲＣから提供された）を１８１培地中（２ｍｌ）で３７℃にて振盪培養した。培養終了後、培養液から菌体を回収し、Ｎｕｃｌｅｏ　Ｓｐｉｎ　Ｔｉｓｓｕｅ（製品名、ＭＡＣＨＥＲＥＹ－ＮＡＧＥＬ社製）を使用してゲノムＤＮＡを抽出した。プライマー名称の末尾に「Ａ」および「Ｂ」を付けたプライマーセット、ならびに、プライマー名称の末尾に「Ｃ」および「Ｄ」を付けたプライマーセット（図５参照）を用いて、相同性領域をＰＣＲ増幅し、断片１および２を得た。反応条件は９８℃（１０ｓｅｃ），５５℃（５ｓｅｃ），７２℃（３０ｓｅｃ），３０ｃｙｃｌｅであった。 (Example B1-a) Preparation of gene disruption plasmid Bacillus pseudofilamus OF4 strain (JCM17055 strain, this strain was provided by Riken BRC through the Ministry of Education, Culture, Sports, Science and Technology National Resource Project) in 181 medium (2 ml) Shaking culture was carried out at 37°C. After the culture was completed, the cells were recovered from the culture medium, and genomic DNA was extracted using Nucleo Spin Tissue (product name, MACHEREY-NAGEL). Using a primer set with "A" and "B" at the end of the primer name and a primer set with "C" and "D" at the end of the primer name (see FIG. 5), the homologous region was identified. Fragments 1 and 2 were obtained by PCR amplification. The reaction conditions were 98° C. (10 sec), 55° C. (5 sec), 72° C. (30 sec), 30 cycles.
　プラスミドｐＡＬ３５１（独立行政法人製品評価技術基盤機構　バイオテクノロジーセンター　特許微生物寄託センター（ＮＰＭＤ）に２０１９年３月１８日付けで寄託されている。受託番号：ＮＩＴＥ　ＢＰ－０２９１８）を、プライマー名称の末尾に「Ｅ」および「Ｆ」を付けたプライマーセット（図５参照）を用いて、ＰＣＲ増幅し、断片３を得た。反応条件は９８℃（１０ｓｅｃ），５５℃（５ｓｅｃ），７２℃（３０ｓｅｃ），３０ｃｙｃｌｅであった。断片３をベクターとして用い、インサートとして断片１、２をライゲーションし、ｐＡＫＮＵ０１～０９を構築した。 Plasmid pAL351 (Deposited on March 18, 2019 at National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary Center (NPMD). Accession number: NITE BP-02918) is added to the end of the primer name. Fragment 3 was obtained by PCR amplification using the primer set labeled “E” and “F” (see FIG. 5). The reaction conditions were 98° C. (10 sec), 55° C. (5 sec), 72° C. (30 sec), 30 cycles. Fragment 3 was used as a vector, and fragments 1 and 2 were ligated as inserts to construct pAKNU01-09.
（実施例Ｂ１－ｂ）形質転換体の取得
　実施例Ｂ１－ａで構築したｐＡＫＮＵ０１～０９をバチルス・シュードフィラマスＡＫＡＬ－００１株（独立行政法人製品評価技術基盤機構　バイオテクノロジーセンター　特許微生物寄託センター（ＮＰＭＤ）に寄託されている。受託番号：ＮＩＴＥ　ＢＰ－０２９２０）に形質転換し、ＡＫＡＬｐ－１０１株～ＡＫＡＬｐ－１０９株を取得した。 (Example B1-b) Acquisition of transformants pAKNU01 to 09 constructed in Example B1-a were transformed into Bacillus pseudofilamus AKAL-001 strain (National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary Center ( NPMD), accession number: NITE BP-02920), and strains AKALp-101 to AKALp-109 were obtained.
（実施例Ｂ１－ｃ）染色体挿入株の取得
　実施例Ｂ１－ｂで取得したＡＫＡＬｐ－１０１株～ＡＫＡＬｐ－１０９株を３０℃にてクロラムフェニコール１０μｇ／ｍＬを含む１８１培地で１日間培養した後、１００倍希釈してクロラムフェニコール１０μｇ／ｍＬを含む１８１培地に塗布し、４３℃にて培養し、プラスミドが染色体に相同組み換えされた染色体挿入株ＡＫＡＬｐ－２０１株～ＡＫＡＬｐ－２０９株を取得した。 (Example B1-c) Acquisition of Chromosome Insertion Strains AKALp-101 to AKALp-109 strains obtained in Example B1-b were cultured at 30° C. for 1 day in 181 medium containing 10 μg/mL chloramphenicol. After that, it was diluted 100-fold and applied to 181 medium containing 10 μg/mL of chloramphenicol, cultured at 43° C., and chromosomally inserted strains AKALp-201 strain to AKALp-209 strain in which the plasmid was homologously recombined into the chromosome. Acquired.
（実施例Ｂ１－ｄ）ｙｊｂＣ遺伝子破壊株の取得
　実施例Ｂ１－ｃで取得したＡＫＡＬｐ－２０１株～ＡＫＡＬｐ－２０９株を３０℃にてクロラムフェニコール１０μｇ／ｍＬを含む１８１培地で１日間培養した後、１００倍希釈して４－クロロフェニルアラニン５ｍＭを含む１８１培地に塗布し、３０℃にて培養し、染色体からプラスミドに加え、それぞれの遺伝子領域が除去された遺伝子破壊株ＡＫＤＮＣ－００１株～ＡＫＤＮＣ－００９株を取得した。遺伝子の欠失は、プライマー名称の末尾に「Ｇ」および「Ｈ」を付けたプライマーセット（図５参照）を用いたＰＣＲにより確認した。反応条件は９８℃（１０ｓｅｃ），５５℃（５ｓｅｃ），７２℃（３０ｓｅｃ），３０ｃｙｃｌｅであった。 (Example B1-d) Acquisition of yjbC gene-disrupted strain The strains AKALp-201 to AKALp-209 obtained in Example B1-c were cultured at 30°C for 1 day in 181 medium containing 10 µg/mL chloramphenicol. After that, it was diluted 100 times and applied to 181 medium containing 5 mM 4-chlorophenylalanine, cultured at 30 ° C., added to the plasmid from the chromosome, and the gene disruption strain AKDNC-001 strain to which each gene region was removed. AKDNC-009 strain was obtained. Gene deletion was confirmed by PCR using a primer set with suffixes "G" and "H" (see FIG. 5). The reaction conditions were 98° C. (10 sec), 55° C. (5 sec), 72° C. (30 sec), 30 cycles.
　構築した菌株と、遺伝子との対応を下記表に示す。 The table below shows the correspondence between the constructed strain and the gene.
Figure JPOXMLDOC01-appb-T000021
Figure JPOXMLDOC01-appb-T000021
実施例Ｂ２：構築した菌株におけるＮ－アセチルカダベリン生産能の評価（ファーメンター培養）
　ＡＫＡＬ－００１株およびＡＫＤＮＣ－００１～００９株を、１８１培地プレート上で、３７℃、１日間培養して、コロニーを形成させた。１８１培地２ｍＬを１４ｍＬ容の試験管に入れ、上記プレートからコロニーを白金耳で植菌し、３７℃で、１８０ｒｐｍで培養を行い、十分な濁度を得るまで培養し、これを本培養のための前培養液とした。 Example B2: Evaluation of N-acetylcadaverine-producing ability in constructed strains (fermenter culture)
Strains AKAL-001 and AKDNC-001 to 009 were cultured on 181 medium plates at 37° C. for 1 day to form colonies. Put 2 mL of 181 medium in a 14 mL test tube, inoculate a colony from the plate with a platinum loop, culture at 37 ° C. and 180 rpm, culture until sufficient turbidity is obtained, and use this for the main culture It was used as a pre-culture solution.
　１００ｍＬ容のジャー培養装置（機種名：Ｂｉｏ　Ｊｒ．８、バイオット社製）に３０ｇ／Ｌグルコースを含むＢＳジャー培地（表Ｂ－３に示す）を入れ、１ｍＬの前培養液を添加し本培養を行った（カダベリン生産試験）。培養条件は、培養温度：３７℃、培養ｐＨ：７．５、アルカリ添加：１０％アンモニア水、攪拌速度：７５０ｒｐｍ、通気速度：０．１ｖｖｍとした。培養中に経時的にサンプリングを行い、培養液中の菌体濁度および培養上清中のジアミン濃度を定量した。培養開始２４ｈ後に５０％グルコース溶液を６ｍｌ添加した。 BS jar medium containing 30 g/L glucose (shown in Table B-3) is placed in a 100 mL jar culture apparatus (model name: Bio Jr. 8, manufactured by Biot), 1 mL of preculture solution is added, and main culture is performed. was performed (cadaverine production test). The culture conditions were culture temperature: 37° C., culture pH: 7.5, addition of alkali: 10% aqueous ammonia, stirring speed: 750 rpm, aeration speed: 0.1 vvm. Sampling was performed over time during the culture, and the cell turbidity in the culture medium and the diamine concentration in the culture supernatant were quantified. 6 ml of 50% glucose solution was added 24 hours after the initiation of culture.
　上記の培養液を、１０，０００ｇ×３分間遠心分離して上清を回収し、培養上清のカダ
ベリン濃度およびＮ－アセチルカダベリン濃度を測定した。具体的には、ＣＧ－１９（ガードカラム）、ＣＳ－１９（分析カラム）（いずれも商品名、サーモフィッシャーサイエンティフィク株式会社製）を連結してイオンクロマト分析（検出器：電気伝導度、カラム温度：３０℃、流速：０．３５ｍＬ／ｍｉｎ、移動相：８ｍＭメタンスルホン酸水溶液→７０ｍＭ　メタンスルホン酸水溶液のグラジエント）を行うことで、培養上清中のカダベリン濃度およびＮ－アセチルカダベリン濃度を定量した。 The above culture medium was centrifuged at 10,000 g×3 minutes, the supernatant was collected, and the cadaverine concentration and N-acetylcadaverine concentration of the culture supernatant were measured. Specifically, CG-19 (guard column) and CS-19 (analytical column) (both trade names, manufactured by Thermo Fisher Scientific Co., Ltd.) are connected to perform ion chromatography analysis (detector: electrical conductivity, Column temperature: 30° C., flow rate: 0.35 mL/min, mobile phase: gradient of 8 mM methanesulfonic acid aqueous solution→70 mM methanesulfonic acid aqueous solution), the concentration of cadaverine and N-acetylcadaverine in the culture supernatant was adjusted. quantified.
Figure JPOXMLDOC01-appb-T000022
Figure JPOXMLDOC01-appb-T000022
　培養４０時間後の菌体密度（ＯＤ）、カダベリンおよびＮ－アセチルカダベリン濃度（ｇ／ｌ）を表Ｂ－４に示す。ＡＫＡＬ－０４３株およびＡＫＤＮＣ－００１～００８株の培養上清からは、カダベリンおよびＮ－アセチルカダベリンが検出された。一方、ＡＫＤＮＣ－００９株の培養上清からはカダベリンは検出されたが、Ｎ－アセチルカダベリンは検出されなかった（表Ｂ－４）。ＡＫＤＮＣ－００９株は、ＡＫＡＬ－０４３株と比較して、カダベリン濃度が１４％増加した。 Table B-4 shows the cell density (OD), cadaverine and N-acetylcadaverine concentrations (g/l) after 40 hours of culture. Cadaverine and N-acetylcadaverine were detected in the culture supernatants of AKAL-043 and AKDNC-001-008 strains. On the other hand, cadaverine was detected in the culture supernatant of the AKDNC-009 strain, but N-acetylcadaverine was not detected (Table B-4). The AKDNC-009 strain had a 14% increase in cadaverine concentration compared to the AKAL-043 strain.
Figure JPOXMLDOC01-appb-T000023
Figure JPOXMLDOC01-appb-T000023
　上記表Ｂ－４の結果について、非変異株であるＡＫＡＬ－００１株よりもアセチルカダベリンの産生量が０．５ｇ／ｌ以上低下した株は、非変異株よりもＮ－アセチルジアミン化合物を生産する活性が野生株と比較し低下していると理解される。 Regarding the results in Table B-4 above, the strains in which the production of acetylcadaverine is 0.5 g/l or more lower than the non-mutant strain AKAL-001 produce more N-acetyldiamine compounds than the non-mutant strain. It is understood that the activity is lowered compared to the wild type.
　本発明は、ジアミンの工業的発酵生産に利用できる。本発明はまた、副生物の生成を抑制し、ジアミンを効率よく製造できるため、本発明の組換え微生物の発酵により、ジアミンの工業的規模での生産に利用できることが期待される。 The present invention can be used for industrial fermentation production of diamine. Since the present invention can also suppress the production of by-products and efficiently produce diamine, it is expected that the fermentation of the recombinant microorganism of the present invention can be used for industrial-scale production of diamine.

Claims (37)

  1. 　ジアミン生産能を有する、好塩性および／または好アルカリ性の組換え微生物であって、
    　該ジアミンが、式：ＮＨ２ＣＨ２(ＣＨ２)ｎＣＨ２ＮＨ２（式中、ｎは０～１０の整数である）で表され、
    　好塩性および／または好アルカリ性の宿主微生物に、ジアミン生産能を有するように改変が行われた、組換え微生物。     A halophilic and/or alkalophilic recombinant microorganism capable of producing diamine,
    The diamine is represented by the formula: NH 2 CH 2 (CH 2 ) n CH 2 NH 2 (wherein n is an integer from 0 to 10),
    A recombinant microorganism in which a halophilic and/or alkalophilic host microorganism has been modified to have diamine-producing ability.
  2. 　前記式中におけるｎが３または４である、請求項１に記載の組換え微生物。 The recombinant microorganism according to claim 1, wherein n in the formula is 3 or 4.
  3. 　Ｌ－リジンデカルボキシラーゼ（ＥＣ　４．１．１．１８）の過剰生産を誘導するように、宿主微生物が１つ以上の遺伝子操作により改変された請求項２に記載の組換え微生物。 The recombinant microorganism according to claim 2, wherein the host microorganism has been modified by one or more genetic manipulations so as to induce overproduction of L-lysine decarboxylase (EC 4.1.1.18).
  4. 　前記遺伝子操作が、下記（Ａ）、（Ｂ）、（Ｃ）、（Ｄ）及び（Ｅ）：
    （Ａ）前記宿主微生物に、前記Ｌ－リジンデカルボキシラーゼをコードする外来性遺伝子を導入する操作、
    （Ｂ）前記宿主微生物内の前記Ｌ－リジンデカルボキシラーゼの内因性遺伝子のコピー数を増加させる操作、
    （Ｃ）前記宿主微生物内の前記Ｌ－リジンデカルボキシラーゼの内因性遺伝子の発現調整領域に変異を導入する操作、
    （Ｄ）前記宿主微生物内の前記Ｌ－リジンデカルボキシラーゼの内因性遺伝子の発現調整領域を、高発現可能な外来調整領域で置換する操作、および
    （Ｅ）前記宿主微生物内の前記Ｌ－リジンデカルボキシラーゼの内因性遺伝子の調整領域を欠失させる操作
    から成る群より選択される１以上の操作である、請求項３に記載の組換え微生物。     The genetic manipulation includes the following (A), (B), (C), (D) and (E):
    (A) an operation of introducing an exogenous gene encoding the L-lysine decarboxylase into the host microorganism;
    (B) increasing the copy number of the endogenous gene for L-lysine decarboxylase in the host microorganism;
    (C) an operation of introducing a mutation into the expression control region of the endogenous gene of the L-lysine decarboxylase in the host microorganism;
    (D) an operation of replacing the expression regulatory region of the endogenous gene of the L-lysine decarboxylase in the host microorganism with an exogenous regulatory region capable of high expression; and (E) the L-lysine decarboxylase in the host microorganism. 4. The recombinant microorganism of claim 3, wherein the manipulation is one or more selected from the group consisting of manipulations that delete the regulatory region of the endogenous gene for carboxylase.
  5. 　配列番号２または配列番号４に示す塩基配列と、８５、９０、９２、９５、９８または９９％以上の相同性を有する塩基配列を含むか、あるいは
    　配列番号１または３に示すアミノ酸配列をコードする塩基配列と８５、９０、９２、９５、９８または９９％以上の相同性を有する塩基配列を含む、請求項２～４のいずれか１項に記載の組換え微生物。     contains a nucleotide sequence having 85, 90, 92, 95, 98 or 99% or more homology with the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, or encodes the amino acid sequence shown in SEQ ID NO: 1 or 3 5. The recombinant microorganism according to any one of claims 2 to 4, comprising a nucleotide sequence having 85, 90, 92, 95, 98 or 99% or more homology with the nucleotide sequence.
  6. 　リジン生産能を向上させるための突然変異操作または遺伝子組換え操作によりさらに改変されている、請求項２または３に記載の組換え微生物。 The recombinant microorganism according to claim 2 or 3, further modified by mutation or gene recombination to improve lysine-producing ability.
  7. 　前記突然変異操作または前記遺伝子組換え操作が、アスパルトキナーゼＩＩＩ（ＥＣ　２．７．２．４）及び４－ヒドロキシ－テトラヒドロジピコリン酸シンターゼ（ＥＣ　４．３．３．７）の少なくとも一方に対するフィードバック阻害を解除する操作である、請求項６に記載の組換え微生物。 The mutagenesis or genetic recombination manipulation feeds back on at least one of aspartokinase III (EC 2.7.2.4) and 4-hydroxy-tetrahydrodipicolinate synthase (EC 4.3.3.7) 7. The recombinant microorganism according to claim 6, which is an operation to release the inhibition.
  8. 　前記宿主微生物が、バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｅｕｄｏｆｉｒｍｕｓ）、バチルス・ハロデュランス（Ｂａｃｉｌｌｕｓ　ｈａｌｏｄｕｒａｎｓ）、及びバチルス・マーマエンシス（Ｂａｃｉｌｌｕｓ　ｍａｒｍａｒｅｎｓｉｓ）からなる群より選択される、請求項１または２に記載の組換え微生物。 3. The combination of claim 1 or 2, wherein said host microorganism is selected from the group consisting of Bacillus pseudofirmus, Bacillus halodurans, and Bacillus marmarensis. replacement microorganisms.
  9. 　前記宿主微生物が、バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｅｕｄｏｆｉｒｍｕｓ）である請求項１または２に記載の組換え微生物。 The recombinant microorganism according to claim 1 or 2, wherein the host microorganism is Bacillus pseudofirmus.
  10. 　請求項１または２に記載の組換え微生物を用いて、式：ＮＨ２ＣＨ２(ＣＨ２)ｎＣＨ２ＮＨ２（式中、ｎは０～１０の整数である）で表されるジアミンを製造する方法。 Using the recombinant microorganism according to claim 1 or 2, a diamine represented by the formula: NH 2 CH 2 (CH 2 ) n CH 2 NH 2 (wherein n is an integer of 0 to 10) How to manufacture.
  11. 　前記式中におけるｎが３または４である、請求項１０に記載の製造方法。 The production method according to claim 10, wherein n in the formula is 3 or 4.
  12. 　前記組換え微生物を５質量％以上の無機塩を含有する培地で培養することによりジアミンを含む培養液を得る培養工程を含む、請求項１０に記載の製造方法。 The production method according to claim 10, comprising a culturing step of obtaining a culture solution containing diamine by culturing the recombinant microorganism in a medium containing 5% by mass or more of an inorganic salt.
  13. 　前記組換え微生物を培養して、菌体を含む培養液を得る培養工程と、
    　前記培養液および／または前記菌体を、５質量％以上の無機塩とリジンとを含有する水溶液と接触させて１，５－ペンタンジアミンを含む反応液を得る反応工程と
    を含む、請求項１０に記載の製造方法。     a culturing step of culturing the recombinant microorganism to obtain a culture solution containing bacterial cells;
    and a reaction step of obtaining a reaction solution containing 1,5-pentanediamine by contacting said culture solution and/or said cells with an aqueous solution containing 5% by mass or more of an inorganic salt and lysine. The manufacturing method described in .
  14. 　前記培養液または反応液から前記組換え微生物を除去する除去工程を含む、請求項１２に記載の製造方法。 The production method according to claim 12, comprising a removal step of removing the recombinant microorganism from the culture solution or reaction solution.
  15. 　前記培養液または反応液を濃縮する濃縮工程を含む、請求項１４に記載の製造方法。 The production method according to claim 14, comprising a concentration step of concentrating the culture solution or reaction solution.
  16. 　前記培養液または反応液のｐＨを１２以上に調整するｐＨ調整工程を含む、請求項１２に記載の製造方法。 The production method according to claim 12, comprising a pH adjustment step of adjusting the pH of the culture solution or reaction solution to 12 or higher.
  17. 　前記培養液または反応液から、ジアミンを含む相と、前記無機塩を含む水相とを相分離する分離工程を含む、請求項１２に記載の製造方法。 The production method according to claim 12, comprising a separation step of phase-separating a phase containing a diamine and an aqueous phase containing the inorganic salt from the culture solution or the reaction solution.
  18. 　前記分離工程において、前記培養液または反応液に有機溶媒を添加することにより、ジアミンおよび有機溶媒を含む相と水相とに相分離する、請求項１２に記載の製造方法。 13. The production method according to claim 12, wherein in the separation step, an organic solvent is added to the culture solution or reaction solution to cause phase separation into a phase containing diamine and an organic solvent and an aqueous phase.
  19. 　前記分離工程において、アルカリ化合物を添加しない、請求項１７に記載の製造方法。 The production method according to claim 17, wherein no alkali compound is added in the separation step.
  20. 　前記無機塩が、炭酸ナトリウム及び／又は硫酸ナトリウムである、請求項１２に記載の製造方法。 The production method according to claim 12, wherein the inorganic salt is sodium carbonate and/or sodium sulfate.
  21. 　前記培養工程及び／又は反応工程により生産したジアミンが、炭酸塩、重炭酸塩、ビス重炭酸塩、カルバミン酸塩、及びビスカルバミン酸塩からなる群より選択される１種以上の形態であり、
    　前記濃縮工程において、前記塩の形態のジアミンを、遊離塩基及び二酸化炭素に転換させ、二酸化炭素を分離させる、請求項１２に記載の製造方法。     The diamine produced by the culture step and/or the reaction step is in one or more forms selected from the group consisting of carbonate, bicarbonate, bisbicarbonate, carbamate, and biscarbamate,
    13. The process according to claim 12, wherein in the concentrating step, the salt form of the diamine is converted into a free base and carbon dioxide, and the carbon dioxide is separated.
  22. 　前記有機溶媒が、ヘキサン、ブタノール、及び２－エチル－１－ヘキサノールからなる群より選択される１種以上である、請求項１８に記載の製造方法。 The production method according to claim 18, wherein the organic solvent is one or more selected from the group consisting of hexane, butanol, and 2-ethyl-1-hexanol.
  23. 　前記ジアミンが、前記組換え微生物による、糖、二酸化炭素、合成ガス、メタノール、及びアミノ酸のうちの少なくとも１つ以上の発酵により生成されたものである、請求項１２に記載の製造方法。 　The production method according to claim 12, wherein the diamine is produced by fermentation of at least one of sugar, carbon dioxide, synthesis gas, methanol, and amino acids by the recombinant microorganism.
  24. 　前記分離した前記無機塩を含む水相を培養液として再使用することを含む、請求項１７に記載の製造方法。 The production method according to claim 17, comprising reusing the separated aqueous phase containing the inorganic salt as a culture solution.
  25. 　得られたジアミンを精製する精製工程を含む、請求項１２に記載の製造方法。 The production method according to claim 12, comprising a purification step of purifying the obtained diamine.
  26. 　ジアミン生産能を有する、好塩性および／または好アルカリ性の組換え微生物であって、 
    　ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成するＮ－アセチル化酵素を抑制する１以上の遺伝子改変を含む、組換え微生物。     A halophilic and/or alkalophilic recombinant microorganism capable of producing diamine,
    A recombinant microorganism comprising one or more genetic modifications that inhibit an N-acetyltransferase that N-acetylates a diamine compound to produce an N-acetyldiamine compound.
  27. 　前記遺伝子改変が、
    ・前記Ｎ－アセチル化酵素をコードする内因性遺伝子の発現を抑制する改変であるか、または、
    ・当該Ｎ－アセチル化酵素の活性が低下する改変である、請求項２６に記載の組換え微生物。     The genetic modification is
    - modification that suppresses the expression of the endogenous gene encoding the N-acetyltransferase, or
    - The recombinant microorganism according to claim 26, wherein the modification reduces the activity of the N-acetyltransferase.
  28. 　Ｎ－アセチルジアミン化合物の生産能が、前記遺伝子改変を含まない非変異株の当該生産能と比較して、抑制されているか又は消失している、請求項２６または２７に記載の組換え微生物。 The recombinant microorganism according to claim 26 or 27, wherein the ability to produce an N-acetyldiamine compound is suppressed or eliminated compared to the production ability of the non-mutant strain containing no genetic modification.
  29. 　前記Ｎ－アセチル化酵素をコードする遺伝子がｙｊｂＣ遺伝子である、請求項２６または２７に記載の組換え微生物。 The recombinant microorganism according to claim 26 or 27, wherein the gene encoding the N-acetyltransferase is the yjbC gene.
  30. 　前記Ｎ－アセチル化酵素が、
    　（Ａ）（Ａ－１）配列番号２３に示されるアミノ酸配列からなるか、
    　（Ａ－２）配列番号２３に示されるアミノ酸配列と８５％以上、９０％以上、９５％以上、９７％以上、９８％以上または９９％以上の配列同一性を有するアミノ酸配列からなり、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するか、もしくは
    　（Ａ－３）配列番号２３に示されるアミノ酸配列に対して１～１０個、１～７個、１～５個、または１～３個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなり、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するか、または
    　（Ｂ）（Ｂ－１）配列番号２４に示される塩基配列からなるＤＮＡ、
    　（Ｂ－２）配列番号２４に示される塩基配列に相補的な塩基配列を有するＤＮＡと緊縮条件下でハイブリダイズし、かつ、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するタンパク質をコードするＤＮＡ、
    　（Ｂ－３）配列番号２４に示される塩基配列と８５％以上、９０％以上、９５％以上、９７％以上、９８％以上または９９％以上の配列同一性を有する塩基配列からなり、かつ、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するタンパク質をコードするＤＮＡ、
    　（Ｂ－４）配列番号２４に示される塩基配列によりコードされるタンパク質のアミノ酸配列に対して１～１０個、１～７個、１～５個、または１～３個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列からなるタンパク質をコードするＤＮＡであって、ジアミン化合物をＮ－アセチル化してＮ－アセチルジアミン化合物を生成する酵素活性を有するタンパク質をコードするＤＮＡ、もしくは
    　（Ｂ－５）配列番号２４に示される塩基配列の縮重異性体からなるＤＮＡ
    にコードされる、請求項２６または２７に記載の組換え微生物。     The N-acetyltransferase is
    (A) (A-1) consists of the amino acid sequence shown in SEQ ID NO: 23,
    (A-2) A diamine compound consisting of an amino acid sequence having 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 23. or (A-3) 1 to 10, 1 to 7, 1 to 5 relative to the amino acid sequence shown in SEQ ID NO: 23 consisting of an amino acid sequence in which 1 or 1 to 3 amino acids are deleted, substituted, inserted and/or added, and having an enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound, or (B) (B-1) DNA consisting of the base sequence shown in SEQ ID NO: 24;
    (B-2) an enzyme that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 24 and N-acetylates a diamine compound to produce an N-acetyldiamine compound DNA encoding a protein having activity;
    (B-3) consists of a base sequence having 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the base sequence shown in SEQ ID NO: 24, and DNA encoding a protein having an enzymatic activity that N-acetylates a diamine compound to produce an N-acetyldiamine compound;
    (B-4) deletion of 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids from the amino acid sequence of the protein encoded by the nucleotide sequence shown in SEQ ID NO: 24; A DNA encoding a protein consisting of a substituted, inserted and/or added amino acid sequence, the DNA encoding a protein having the enzymatic activity of N-acetylating a diamine compound to produce an N-acetyldiamine compound, or B-5) DNA consisting of a degenerate isomer of the base sequence shown in SEQ ID NO: 24
    28. The recombinant microorganism of claim 26 or 27, encoded by
  31. 　好塩性および／または好アルカリ性の宿主微生物に対して、前記Ｎ－アセチル化酵素を抑制する１以上の遺伝子改変が行われた、請求項２６または２７に記載の組換え微生物。 The recombinant microorganism according to claim 26 or 27, wherein the halophilic and/or alkalophilic host microorganism has been subjected to one or more genetic modifications that inhibit said N-acetyltransferase.
  32. 　前記宿主微生物が、バチルス・シュードフィラマス（Ｂａｃｉｌｌｕｓ　ｐｓｅｕｄｏｆｉｒｍｕｓ）、バチルス・ハロデュランス(Ｂａｃｉｌｌｕｓ　ｈａｌｏｄｕｒａｎｓ)およびバチルス・マーマエンシス(Ｂａｃｉｌｌｕｓ　ｍａｒｍａｒｅｎｓｉｓ)からなる群より選択される、請求項２６または２７に記載の組換え微生物。 28. The recombinant of claim 26 or 27, wherein said host microorganism is selected from the group consisting of Bacillus pseudofirmus, Bacillus halodurans and Bacillus marmarensis. microbes.
  33. 　前記ジアミン化合物が、式：ＮＨ２ＣＨ２(ＣＨ２)ｎＣＨ２ＮＨ２（式中、ｎは、０、１、２、３、４、５、６、７、８、９または１０である。）で表される、請求項２６または２７に記載の組換え微生物。 The diamine compound has the formula: NH2CH2 ( CH2 )nCH2NH2, where n is 0, 1, 2 , 3, 4 , 5, 6 , 7, 8, 9 or 10 28. The recombinant microorganism according to claim 26 or 27, represented by .
  34. 　前記ジアミン化合物がカダベリンである、請求項２６または２７に記載の組換え微生物。 The recombinant microorganism according to claim 26 or 27, wherein said diamine compound is cadaverine.
  35. 　請求項２６または２７に記載の組換え微生物を培養して、当該組み換え微生物の培養物および／または培養物の抽出物を得る培養工程を含む、ジアミン化合物の製造方法。 A method for producing a diamine compound, comprising a culturing step of culturing the recombinant microorganism according to claim 26 or 27 to obtain a culture of the recombinant microorganism and/or an extract of the culture.
  36. 　前記培養物および／または前記培養物の抽出物を、基質化合物と混合して混合液を得る混合工程をさらに含む、請求項３５に記載の製造方法。 The production method according to claim 35, further comprising a mixing step of mixing the culture and/or the extract of the culture with a substrate compound to obtain a mixed solution.
  37. 　前記培養物または前記混合液から、ジアミン化合物を回収する回収工程をさらに含む、請求項３５に記載の製造方法。 The production method according to claim 35, further comprising a recovery step of recovering the diamine compound from the culture or the mixed solution.
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